Antibody drug conjugates

ABSTRACT

The present invention relates to anti-cKIT antibodies, antibody fragments, antibody drug conjugates, and their uses for the treatment of cancer.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to provisional application Ser. No.61/793,641, filed Mar. 15, 2013.

FIELD OF THE INVENTION

The present disclosure is directed to anti-cKIT antibodies, antibodyfragments, antibody drug conjugates, and their uses for the treatment ofcancer.

BACKGROUND OF THE INVENTION

cKIT is a single transmembrane, receptor tyrosine kinase that binds theligand Stem Cell Factor (SCF). SCF induces homodimerization of cKITwhich activates its tyrosine kinase activity and signals through boththe PI3-AKT and MAPK pathways (Kindblom et al., Am J. Path. 1998152(5):1259). cKIT was initially discovered as an oncogene as atruncated form expressed by a feline retrovirus (Besmer et al., 1986).Cloning of the corresponding human gene demonstrated that cKIT is amember of the type III class of receptor tyrosine kinases, which countamong the family members; FLT3,CSF-1 receptor and PDGF receptor.

Mice that are mutant for cKIT have shown that cKIT is required for thedevelopment of hematopoietic cells, germ cells, mast cells andmelanocytes. In the human, cKIT loss of function may lead to deafnessand de-pigmentation of the skin and hair. A number of gain of functionmutations for cKIT have been described in various cancers. Such cancersinclude gastro-intestinal-stromal tumors (GIST), acute myeloid leukemia(AML), small cell lung cancer (SCLC), mast cell leukemia (MCL) andpancreatic cancer (Hirota et al., Science 1998 (279):577; Esposito etal., Lab. Invets. 2002 82(11):1481).

Because of these preliminary indications that cKIT was an oncogene, anantibody was generated that identified cKIT as a marker of AML (Gadd etal., Leuk. Res. 1985 (9):1329). This murine monoclonal, known as YB5.8B,was generated by using leukemic blast cells from a human patient andbound cKIT, which was abundantly expressed on the surface of the AMLcells, but did not detect cKIT on normal blood or bone marrow cells(Gadd et al., supra). A second cKIT antibody (SR-1) was generated thatblocked the binding of SCF to cKIT and thus blocked cKIT signaling(Broudy et al., Blood 1992 79(2):338). The biological effect of the SR-1antibody was to inhibit BFU-E and CFU-GM growth, and based on thisevidence, suggested using it for further studies on hematopoiesis ortumor cell growth (Broudy et al., supra).

In further cancer studies, investigators found that treatment withImatinib, a small molecule inhibitor of cKIT, would significantly reduceproliferation of GIST cell lines. However, Imatinib treated cells becomeresistant over time due to secondary mutations in cKIT (Edris et al.,Proc. Nat. Acad. Sci. USA, Early On-line Edition 2013). However, if theGIST cells were treated with the SR-1 antibody as a second therapeutic,there was significant decrease in cell proliferation, and a decrease incKIT expression on the cell surface (Edris et al., supra). Thus, a nakedSR-1 antibody was efficacious in addressing the problem of Imatinibresistance in human GIST lines, suggesting that an Imatinib/anti-cKITantibody combination may be useful.

Antibody Drug Conjugates

Antibody drug conjugates (“ADCs”) have been used for the local deliveryof cytotoxic agents in the treatment of cancer (see e.g., Lambert, Cuff.Opinion In Pharmacology 5:543-549, 2005). ADCs allow targeted deliveryof the drug moiety where maximum efficacy with minimal toxicity may beachieved. As more ADCs show promising clinical results, there is anincreased need to develop new therapeutics for cancer therapy.

SUMMARY OF THE INVENTION

The present disclosure is directed to an antibody drug conjugate of theformula Ab-(L-(D)_(m))_(n) or a pharmaceutically acceptable saltthereof; wherein Ab is an antibody or antigen binding fragment thereofthat specifically binds to an epitope of human cKIT; L is a linker; D isa drug moiety; m is an integer from 1 to 8; and n is an integer from 1to 10.

The antibody drug conjugate wherein said n is 3 or 4.

The antibody drug conjugate, wherein said antibody or antigen bindingfragment thereof specifically binds the extracellular domain of cKIT(SEQ ID NO.160).

The antibody drug conjugate, wherein said antibody or antigen bindingfragment specifically binds to an epitope of human cKIT at domains 1-3(SEQ ID NO.155).

The antibody drug conjugate, wherein said antibody or antigen bindingfragment thereof specifically binds human cKIT at SEQ ID NO. 161 or SEQID NO.162.

The antibody drug conjugate wherein said antibody or antigen bindingfragment thereof comprises a heavy chain variable region that comprises:(a) a VH CDR1 of SEQ ID NO: 3, (b) a VH CDR2 of SEQ ID NO: 4, (c) a VHCDR3 of SEQ ID NO: 5; and a light chain variable region that comprises:(d) a VL CDR1 of SEQ ID NO: 12, (e) a VL CDR2 of SEQ ID NO: 13, and (f)a VL CDR3 of SEQ ID NO: 14.

The antibody drug conjugate wherein said antibody or antigen bindingfragment thereof comprises a heavy chain variable region that comprises:(a) a VH CDR1 of SEQ ID NO: 22, (b) a VH CDR2 of SEQ ID NO: 23, (c) a VHCDR3 of SEQ ID NO: 24; and a light chain variable region that comprises:(d) a VL CDR1 of SEQ ID NO: 31, (e) a VL CDR2 of SEQ ID NO: 32, and (f)a VL CDR3 of SEQ ID NO: 33.

The antibody drug conjugate wherein said antibody or antigen bindingfragment thereof comprises a heavy chain variable region that comprises:(a) a VH CDR1 of SEQ ID NO: 40, (b) a VH CDR2 of SEQ ID NO: 41, (c) a VHCDR3 of SEQ ID NO: 42; and a light chain variable region that comprises:(d) a VL CDR1 of SEQ ID NO: 49, (e) a VL CDR2 of SEQ ID NO: 50, and (f)a VL CDR3 of SEQ ID NO: 51.

The antibody drug conjugate wherein said antibody or antigen bindingfragment thereof comprises a heavy chain variable region that comprises:(a) a VH CDR1 of SEQ ID NO: 58, (b) a VH CDR2 of SEQ ID NO: 59, (c) a VHCDR3 of SEQ ID NO: 60; and a light chain variable region that comprises:(d) a VL CDR1 of SEQ ID NO: 67, (e) a VL CDR2 of SEQ ID NO: 68, and (f)a VL CDR3 of SEQ ID NO: 69.

The antibody drug conjugate wherein said antibody or antigen bindingfragment thereof comprises a heavy chain variable region that comprises:(a) a VH CDR1 of SEQ ID NO: 76, (b) a VH CDR2 of SEQ ID NO: 77, (c) a VHCDR3 of SEQ ID NO: 78; and a light chain variable region that comprises:(d) a VL CDR1 of SEQ ID NO: 85, (e) a VL CDR2 of SEQ ID NO: 86, and (f)a VL CDR3 of SEQ ID NO: 87.

The antibody drug conjugate wherein said antibody or antigen bindingfragment thereof comprises a heavy chain variable region that comprises:(a) a VH CDR1 of SEQ ID NO: 94, (b) a VH CDR2 of SEQ ID NO: 95, (c) a VHCDR3 of SEQ ID NO: 96; and a light chain variable region that comprises:(d) a VL CDR1 of SEQ ID NO: 103, (e) a VL CDR2 of SEQ ID NO: 104, and(f) a VL CDR3 of SEQ ID NO: 105.

The antibody drug conjugate wherein said antibody or antigen bindingfragment thereof comprises a heavy chain variable region that comprises:(a) a VH CDR1 of SEQ ID NO: 112, (b) a VH CDR2 of SEQ ID NO: 113, (c) aVH CDR3 of SEQ ID NO: 114; and a light chain variable region thatcomprises: (d) a VL CDR1 of SEQ ID NO: 121, (e) a VL CDR2 of SEQ ID NO:122, and (f) a VL CDR3 of SEQ ID NO: 123.

The antibody drug conjugate wherein said antibody or antigen bindingfragment thereof comprises a heavy chain variable region that comprises:(a) a VH CDR1 of SEQ ID NO: 130, (b) a VH CDR2 of SEQ ID NO: 131, (c) aVH CDR3 of SEQ ID NO: 132; and a light chain variable region thatcomprises: (d) a VL CDR1 of SEQ ID NO: 139, (e) a VL CDR2 of SEQ ID NO:140, and (f) a VL CDR3 of SEQ ID NO: 141.

The antibody drug conjugate wherein said linker is selected from thegroup consisting of a cleavable linker, a non-cleavable linker, ahydrophilic linker, a procharged linker and a dicarboxylic acid basedlinker.

The antibody drug conjugate wherein the linker is derived from across-linking reagent selected from the group consisting ofN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl4-(2-pyridyldithio)butanoate (SPDB),N-succinimidyl-4-(2-pyridyldithio)-2-sulfo-butanoate (sulfo-SPDB),N-succinimidyl iodoacetate (SIA),N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), maleimide PEG NHS,N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC),N-sulfosuccinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate(sulfo-SMCC) or 2,5-dioxopyrrolidin-1-yl17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazaheptadecan-1-oate(CX1-1).

The antibody drug conjugate wherein said linker is derived from thecross-linking reagent N-succinimidyl4-(maleimidomethyl)cyclohexanecarboxylate (SMCC).

The antibody drug conjugate wherein the cytotoxic agent is amaytansinoid.

The antibody drug conjugate wherein the maytansinoid isN(2′)-deacetyl-N(2′)-(3-mercapto-1-oxopropyl)-maytansine (DM1) orN(2′)-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4).

An antibody drug conjugate of the formula

or a pharmaceutically acceptable salt thereof; wherein Ab is an antibodyor antigen binding fragment thereof that specifically binds to humancKIT, and comprises at least n number of primary amines; and n is aninteger from 1 to 10.

The antibody drug conjugate wherein said Ab is an antibody or antigenbinding fragment thereof comprises a heavy chain variable region thatcomprises: (a) a VH CDR1 of SEQ ID NO: 3, (b) a VH CDR2 of SEQ ID NO: 4,(c) a VH CDR3 of SEQ ID NO: 5; and a light chain variable region thatcomprises: (d) a VL CDR1 of SEQ ID NO: 12, (e) a VL CDR2 of SEQ ID NO:13, and (f) a VL CDR3 of SEQ ID NO: 14.

The antibody drug conjugate wherein said Ab is an antibody or antigenbinding fragment thereof comprises a heavy chain variable region thatcomprises: (a) a VH CDR1 of SEQ ID NO: 22, (b) a VH CDR2 of SEQ ID NO:23, (c) a VH CDR3 of SEQ ID NO: 24; and a light chain variable regionthat comprises: (d) a VL CDR1 of SEQ ID NO: 31, (e) a VL CDR2 of SEQ IDNO: 32, and (f) a VL CDR3 of SEQ ID NO: 33.

The antibody drug conjugate wherein said Ab is an antibody or antigenbinding fragment thereof comprises a heavy chain variable region thatcomprises: (a) a VH CDR1 of SEQ ID NO: 40, (b) a VH CDR2 of SEQ ID NO:41, (c) a VH CDR3 of SEQ ID NO: 42; and a light chain variable regionthat comprises: (d) a VL CDR1 of SEQ ID NO: 49, (e) a VL CDR2 of SEQ IDNO: 50, and (f) a VL CDR3 of SEQ ID NO: 51.

The antibody drug conjugate wherein said Ab is an antibody or antigenbinding fragment thereof comprises a heavy chain variable region thatcomprises: (a) a VH CDR1 of SEQ ID NO: 58, (b) a VH CDR2 of SEQ ID NO:59, (c) a VH CDR3 of SEQ ID NO: 60; and a light chain variable regionthat comprises: (d) a VL CDR1 of SEQ ID NO: 67, (e) a VL CDR2 of SEQ IDNO: 68, (f) a VL CDR3 of SEQ ID NO: 69.

The antibody drug conjugate wherein said Ab is an antibody or antigenbinding fragment thereof comprises a heavy chain variable region thatcomprises: (a) a VH CDR1 of SEQ ID NO: 76, (b) a VH CDR2 of SEQ ID NO:77, (c) a VH CDR3 of SEQ ID NO: 78; and a light chain variable regionthat comprises: (d) a VL CDR1 of SEQ ID NO: 85, (e) a VL CDR2 of SEQ IDNO: 86, and (f) a VL CDR3 of SEQ ID NO: 87.

The antibody drug conjugate wherein said Ab is an antibody or antigenbinding fragment thereof comprises a heavy chain variable region thatcomprises: (a) a VH CDR1 of SEQ ID NO: 94, (b) a VH CDR2 of SEQ ID NO:95, (c) a VH CDR3 of SEQ ID NO: 96; and a light chain variable regionthat comprises: (d) a VL CDR1 of SEQ ID NO: 103, (e) a VL CDR2 of SEQ IDNO: 104, and (f) a VL CDR3 of SEQ ID NO: 105.

The antibody drug conjugate wherein said Ab is an antibody or antigenbinding fragment thereof comprises a heavy chain variable region thatcomprises: (a) a VH CDR1 of SEQ ID NO: 112, (b) a VH CDR2 of SEQ ID NO:113, (c) a VH CDR3 of SEQ ID NO: 114; and a light chain variable regionthat comprises: (d) a VL CDR1 of SEQ ID NO: 121, (e) a VL CDR2 of SEQ IDNO: 122, and (f) a VL CDR3 of SEQ ID NO: 123.

The antibody drug conjugate wherein said Ab is an antibody or antigenbinding fragment thereof comprises a heavy chain variable region thatcomprises: (a) a VH CDR1 of SEQ ID NO: 130, (b) a VH CDR2 of SEQ ID NO:131, (c) a VH CDR3 of SEQ ID NO: 132; and a light chain variable regionthat comprises: (d) a VL CDR1 of SEQ ID NO: 139, (e) a VL CDR2 of SEQ IDNO: 140, and (f) a VL CDR3 of SEQ ID NO: 141.

The antibody drug conjugate wherein said m is 1.

The antibody drug conjugate wherein said n is 3 or 4.

The antibody drug conjugate wherein said antibody is a human antibody.

The antibody drug conjugate wherein said antibody is a monoclonalantibody.

The antibody drug conjugate wherein said linker is selected from thegroup consisting of a cleavable linker, a non-cleavable linker, ahydrophilic linker, a procharged linker and a dicarboxylic acid basedlinker.

The antibody drug conjugate wherein the linker is derived from across-linking reagent selected from the group consisting ofN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl4-(2-pyridyldithio)butanoate (SPDB),N-succinimidyl-4-(2-pyridyldithio)-2-sulfo-butanoate (sulfo-SPDB),N-succinimidyl iodoacetate (SIA),N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), maleimide PEG NHS,N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC),N-sulfosuccinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate(sulfo-SMCC) or 2,5-dioxopyrrolidin-1-yl17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazaheptadecan-1-oate(CX1-1).

The antibody drug conjugate wherein said linker is N-succinimidyl4-(maleimidomethyl)cyclohexanecarboxylate (SMCC).

The antibody drug conjugate wherein said drug moiety is selected from agroup consisting of a V-ATPase inhibitor, a pro-apoptotic agent, a Bcl2inhibitor, an MCL1 inhibitor, a HSP90 inhibitor, an IAP inhibitor, anmTor inhibitor, a microtubule stabilizer, a microtubule destabilizer, anauristatin, a dolastatin, a maytansinoid, a MetAP (methionineaminopeptidase), an inhibitor of nuclear export of proteins CRM1, aDPPIV inhibitor, proteasome inhibitors, inhibitors of phosphoryltransfer reactions in mitochondria, a protein synthesis inhibitor, akinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a kinesininhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylatingagent, a DNA intercalator, a DNA minor groove binder and a DHFRinhibitor.

The antibody drug conjugate wherein the cytotoxic agent is amaytansinoid.

The antibody drug conjugate wherein the maytansinoid isN(2′)-deacetyl-N(2′)-(3-mercapto-1-oxopropyl)-maytansine (DM1) orN(2′)-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4).

A pharmaceutical composition comprising the antibody drug conjugate ofany one of the antibody drug conjugates herein and a pharmaceuticallyacceptable carrier.

The pharmaceutical composition wherein said composition is prepared as alyophilisate.

The pharmaceutical composition wherein said lyophilisate comprises theantibody drug conjugate, sodium succinate, and polysorbate 20.

A method of treating an cKIT positive cancer in a patient in needthereof, comprising administering to said patient any of antibody drugconjugates or pharmaceutical compositions disclosed herein.

The method wherein said cancer is selected from the group consisting ofgastrointestinal stromal tumors (GIST), small cell lung cancer (SCLC),acute myelod leukemia (AML), melanoma, mast cell leukemia (MCL),mastocytosis, neurofibromatosis, breast cancer, non-small cell lungcancer (NSCLC) and pancreatic cancer.

An antibody drug conjugate for use as a medicament.

An antibody drug conjugate or pharmaceutical composition for use in thetreatment of a cKIT positive cancer.

The antibody or antigen binding fragment wherein said antibody orantigen binding fragment is a single chain antibody (scFv).

A nucleic acid that encodes the antibody or antigen binding fragment.

A vector comprising the nucleic acid.

A host cell comprising the vector.

A process for producing an antibody or antigen binding fragmentcomprising cultivating the host cell and recovering the antibody fromthe culture.

A process for producing an anti-cKIT antibody drug conjugate comprising:(a) chemically linking SMCC to a drug moiety DM-1; (b) conjugating saidlinker-drug to the antibody recovered from the cell culture of claim 49;and (c) purifying the antibody drug conjugate.

The antibody drug conjugate having an average maytansinoid to antibodyratio (MAR), measured with a UV spectrophotometer, of about 3.5.

A diagnostic reagent comprising the antibody or antigen binding fragmentthereof which is labeled.

The diagnostic reagent wherein the label is selected from the groupconsisting of a radiolabel, a fluorophore, a chromophore, an imagingagent, and a metal ion.

DEFINITIONS

Unless stated otherwise, the following terms and phrases as used hereinare intended to have the following meanings:

The term “alkyl” refers to a monovalent saturated hydrocarbon chainhaving the specified number of carbon atoms. For example, C₁₋₆ alkylrefers to an alkyl group having from 1 to 6 carbon atoms. Alkyl groupsmay be straight or branched. Representative branched alkyl groups haveone, two, or three branches. Examples of alkyl groups include, but arenot limited to, methyl, ethyl, propyl (n-propyl and isopropyl), butyl(n-butyl, isobutyl, sec-butyl, and t-butyl), pentyl (n-pentyl,isopentyl, and neopentyl), and hexyl.

The term “antibody” as used herein refers to a polypeptide of theimmunoglobulin family that is capable of binding a corresponding antigennon-covalently, reversibly, and in a specific manner. For example, anaturally occurring IgG antibody is a tetramer comprising at least twoheavy (H) chains and two light (L) chains inter-connected by disulfidebonds. Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as VH) and a heavy chain constant region. The heavychain constant region is comprised of three domains, CH1, CH2 and CH3.Each light chain is comprised of a light chain variable region(abbreviated herein as VL) and a light chain constant region. The lightchain constant region is comprised of one domain, CL. The VH and VLregions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). Each VHand VL is composed of three CDRs and four FRs arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy andlight chains contain a binding domain that interacts with an antigen.The constant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (C1q)of the classical complement system.

The term “antibody” includes, but is not limited to, monoclonalantibodies, human antibodies, humanized antibodies, camelid antibodies,chimeric antibodies, and anti-idiotypic (anti-Id) antibodies (including,e.g., anti-Id antibodies to antibodies of the present disclosure). Theantibodies can be of any isotype/class (e.g., IgG, IgE, IgM, IgD, IgAand IgY), or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2).

“Complementarity-determining domains” or “complementary-determiningregions (“CDRs”) interchangeably refer to the hypervariable regions ofVL and VH. The CDRs are the target protein-binding site of the antibodychains that harbors specificity for such target protein. There are threeCDRs (CDR1-3, numbered sequentially from the N-terminus) in each humanVL or VH, constituting about 15-20% of the variable domains. The CDRsare structurally complementary to the epitope of the target protein andare thus directly responsible for the binding specificity. The remainingstretches of the VL or VH, the so-called framework regions, exhibit lessvariation in amino acid sequence (Kuby, Immunology, 4th ed., Chapter 4.W.H. Freeman & Co., New York, 2000).

The positions of the CDRs and framework regions can be determined usingvarious well known definitions in the art, e.g., Kabat, Chothia, and AbM(see, e.g., Johnson et al., Nucleic Acids Res., 29:205-206 (2001);Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al.,Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol., 227:799-817(1992); Al-Lazikani et al., J. Mol. Biol., 273:927-748 (1997)).Definitions of antigen combining sites are also described in thefollowing: Ruiz et al., Nucleic Acids Res., 28:219-221 (2000); andLefranc, M. P., Nucleic Acids Res., 29:207-209 (2001); MacCallum et al.,J. Mol. Biol., 262:732-745 (1996); and Martin et al., Proc. Natl. Acad.Sci. USA, 86:9268-9272 (1989); Martin et al., Methods Enzymol.,203:121-153 (1991); and Rees et al., In Sternberg M. J. E. (ed.),Protein Structure Prediction, Oxford University Press, Oxford, 141-172(1996).

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the light (VL) and heavy (VH) chain portions determineantigen recognition and specificity. Conversely, the constant domains ofthe light chain (CL) and the heavy chain (CH1, CH2 or CH3) conferimportant biological properties such as secretion, transplacentalmobility, Fc receptor binding, complement binding, and the like. Byconvention, the numbering of the constant region domains increases asthey become more distal from the antigen binding site or amino-terminusof the antibody. The N-terminus is a variable region and at theC-terminus is a constant region; the CH3 and CL domains actuallycomprise the carboxy-terminal domains of the heavy and light chain,respectively.

The term “antigen binding fragment”, as used herein, refers to one ormore portions of an antibody that retain the ability to specificallyinteract with (e.g., by binding, steric hindrance,stabilizing/destabilizing, spatial distribution) an epitope of anantigen. Examples of binding fragments include, but are not limited to,single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), Fab fragments,F(ab′) fragments, a monovalent fragment consisting of the VL, VH, CL andCH1 domains; a F(ab)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; a Fdfragment consisting of the VH and CH1 domains; a Fv fragment consistingof the VL and VH domains of a single arm of an antibody; a dAb fragment(Ward et al., Nature 341:544-546, 1989), which consists of a VH domain;and an isolated complementarity determining region (CDR), or otherepitope-binding fragments of an antibody.

Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules (known as single chain Fv (“scFv”); see, e.g., Bird et al.,Science 242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci.85:5879-5883, 1988). Such single chain antibodies are also intended tobe encompassed within the term “antigen binding fragment.” These antigenbinding fragments are obtained using conventional techniques known tothose of skill in the art, and the fragments are screened for utility inthe same manner as are intact antibodies.

Antigen binding fragments can also be incorporated into single domainantibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies,triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger andHudson, Nature Biotechnology 23:1126-1136, 2005). Antigen bindingfragments can be grafted into scaffolds based on polypeptides such asfibronectin type 111 (Fn3) (see U.S. Pat. No. 6,703,199, which describesfibronectin polypeptide monobodies).

Antigen binding fragments can be incorporated into single chainmolecules comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which,together with complementary light chain polypeptides, form a pair ofantigen binding regions (Zapata et al., Protein Eng. 8:1057-1062, 1995;and U.S. Pat. No. 5,641,870).

The term “monoclonal antibody” or “monoclonal antibody composition” asused herein refers to polypeptides, including antibodies and antigenbinding fragments that have substantially identical amino acid sequenceor are derived from the same genetic source. This term also includespreparations of antibody molecules of single molecular composition. Amonoclonal antibody composition displays a single binding specificityand affinity for a particular epitope.

The term “human antibody”, as used herein, includes antibodies havingvariable regions in which both the framework and CDR regions are derivedfrom sequences of human origin. Furthermore, if the antibody contains aconstant region, the constant region also is derived from such humansequences, e.g., human germline sequences, or mutated versions of humangermline sequences or antibody containing consensus framework sequencesderived from human framework sequences analysis, for example, asdescribed in Knappik et al., J. Mol. Biol. 296:57-86, 2000).

The human antibodies of the present disclosure can include amino acidresidues not encoded by human sequences (e.g., mutations introduced byrandom or site-specific mutagenesis in vitro or by somatic mutation invivo, or a conservative substitution to promote stability ormanufacturing).

The term “recognize” as used herein refers to an antibody or antigenbinding fragment thereof that finds and interacts (e.g., binds) with itsepitope, whether that epitope is linear or conformational. The term“epitope” refers to a site on an antigen to which an antibody or antigenbinding fragment of the disclosure specifically binds. Epitopes can beformed both from contiguous amino acids or noncontiguous amino acidsjuxtaposed by tertiary folding of a protein. Epitopes formed fromcontiguous amino acids are typically retained on exposure to denaturingsolvents, whereas epitopes formed by tertiary folding are typically loston treatment with denaturing solvents. An epitope typically includes atleast 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in aunique spatial conformation. Methods of determining spatial conformationof epitopes include techniques in the art, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance (see, e.g.,Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996)).

The phrase “specifically binds” or “selectively binds,” when used in thecontext of describing the interaction between an antigen (e.g., aprotein) and an antibody, antibody fragment, or antibody-derived bindingagent, refers to a binding reaction that is determinative of thepresence of the antigen in a heterogeneous population of proteins andother biologics, e.g., in a biological sample, e.g., a blood, serum,plasma or tissue sample. Thus, under certain designated immunoassayconditions, the antibodies or binding agents with a particular bindingspecificity bind to a particular antigen at least two times thebackground and do not substantially bind in a significant amount toother antigens present in the sample. In one aspect, under designatedimmunoassay conditions, the antibody or binding agent with a particularbinding specificity binds to a particular antigen at least ten (10)times the background and does not substantially bind in a significantamount to other antigens present in the sample. Specific binding to anantibody or binding agent under such conditions may require the antibodyor agent to have been selected for its specificity for a particularprotein. As desired or appropriate, this selection may be achieved bysubtracting out antibodies that cross-react with molecules from otherspecies (e.g., mouse or rat) or other subtypes. Alternatively, in someaspects, antibodies or antibody fragments are selected that cross-reactwith certain desired molecules.

The term “affinity” as used herein refers to the strength of interactionbetween antibody and antigen at single antigenic sites. Within eachantigenic site, the variable region of the antibody “arm” interactsthrough weak non-covalent forces with antigen at numerous sites; themore interactions, the stronger the affinity.

The term “isolated antibody” refers to an antibody that is substantiallyfree of other antibodies having different antigenic specificities. Anisolated antibody that specifically binds to one antigen may, however,have cross-reactivity to other antigens. Moreover, an isolated antibodymay be substantially free of other cellular material and/or chemicals.

The term “corresponding human germline sequence” refers to the nucleicacid sequence encoding a human variable region amino acid sequence orsubsequence that shares the highest determined amino acid sequenceidentity with a reference variable region amino acid sequence orsubsequence in comparison to all other all other known variable regionamino acid sequences encoded by human germline immunoglobulin variableregion sequences. The corresponding human germline sequence can alsorefer to the human variable region amino acid sequence or subsequencewith the highest amino acid sequence identity with a reference variableregion amino acid sequence or subsequence in comparison to all otherevaluated variable region amino acid sequences. The corresponding humangermline sequence can be framework regions only, complementaritydetermining regions only, framework and complementary determiningregions, a variable segment (as defined above), or other combinations ofsequences or subsequences that comprise a variable region. Sequenceidentity can be determined using the methods described herein, forexample, aligning two sequences using BLAST, ALIGN, or another alignmentalgorithm known in the art. The corresponding human germline nucleicacid or amino acid sequence can have at least about 90%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the referencevariable region nucleic acid or amino acid sequence.

A variety of immunoassay formats may be used to select antibodiesspecifically immunoreactive with a particular protein. For example,solid-phase ELISA immunoassays are routinely used to select antibodiesspecifically immunoreactive with a protein (see, e.g., Harlow & Lane,Using Antibodies, A Laboratory Manual (1998), for a description ofimmunoassay formats and conditions that can be used to determinespecific immunoreactivity). Typically a specific or selective bindingreaction will produce a signal at least twice over the background signaland more typically at least 10 to 100 times over the background.

The term “equilibrium dissociation constant (KD, M)” refers to thedissociation rate constant (kd, time−1) divided by the association rateconstant (ka, time−1, M−1). Equilibrium dissociation constants can bemeasured using any known method in the art. The antibodies of thepresent disclosure generally will have an equilibrium dissociationconstant of less than about 10⁻⁷ or 10⁻⁸ M, for example, less than about10⁻⁹ M or 10⁻¹⁰ M, in some aspects, less than about 10⁻¹¹ M, 10⁻¹² M or10⁻¹³ M.

The term “bioavailability” refers to the systemic availability (i.e.,blood/plasma levels) of a given amount of drug administered to apatient. Bioavailability is an absolute term that indicates measurementof both the time (rate) and total amount (extent) of drug that reachesthe general circulation from an administered dosage form.

As used herein, the phrase “consisting essentially of” refers to thegenera or species of active pharmaceutical agents included in a methodor composition, as well as any excipients inactive for the intendedpurpose of the methods or compositions. In some aspects, the phrase“consisting essentially of” expressly excludes the inclusion of one ormore additional active agents other than an antibody drug conjugate ofthe present disclosure. In some aspects, the phrase “consistingessentially of” expressly excludes the inclusion of one or moreadditional active agents other than an antibody drug conjugate of thepresent disclosure and a second co-administered agent.

The term “amino acid” refers to naturally occurring, synthetic, andunnatural amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally occurring amino acids are those encoded by thegenetic code, as well as those amino acids that are later modified,e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine Amino acidanalogs refer to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α-carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

The term “conservatively modified variant” applies to both amino acidand nucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidthat encodes a polypeptide is implicit in each described sequence.

For polypeptide sequences, “conservatively modified variants” includeindividual substitutions, deletions or additions to a polypeptidesequence which result in the substitution of an amino acid with achemically similar amino acid. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles. Thefollowing eight groups contain amino acids that are conservativesubstitutions for one another: 1) Alanine (A), Glycine (G); 2) Asparticacid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4)Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine(M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7)Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see,e.g., Creighton, Proteins (1984)). In some aspects, the term“conservative sequence modifications” are used to refer to amino acidmodifications that do not significantly affect or alter the bindingcharacteristics of the antibody containing the amino acid sequence.

The term “optimized” as used herein refers to a nucleotide sequence thathas been altered to encode an amino acid sequence using codons that arepreferred in the production cell or organism, generally a eukaryoticcell, for example, a yeast cell, a Pichia cell, a fungal cell, aTrichoderma cell, a Chinese Hamster Ovary cell (CHO) or a human cell.The optimized nucleotide sequence is engineered to retain completely oras much as possible the amino acid sequence originally encoded by thestarting nucleotide sequence, which is also known as the “parental”sequence.

The terms “percent identical” or “percent identity,” in the context oftwo or more nucleic acids or polypeptide sequences, refers to the extentto which two or more sequences or subsequences that are the same. Twosequences are “identical” if they have the same sequence of amino acidsor nucleotides over the region being compared. Two sequences are“substantially identical” if two sequences have a specified percentageof amino acid residues or nucleotides that are the same (i.e., 60%identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identityover a specified region, or, when not specified, over the entiresequence), when compared and aligned for maximum correspondence over acomparison window, or designated region as measured using one of thefollowing sequence comparison algorithms or by manual alignment andvisual inspection. Optionally, the identity exists over a region that isat least about 30 nucleotides (or 10 amino acids) in length, or morepreferably over a region that is 100 to 500 or 1000 or more nucleotides(or 20, 50, 200 or more amino acids) in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith and Waterman, Adv. Appl. Math. 2:482c (1970), by the homologyalignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443(1970), by the search for similarity method of Pearson and Lipman, Proc.Natl. Acad. Sci. USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), or by manual alignment and visual inspection (see, e.g.,Brent et al., Current Protocols in Molecular Biology, 2003).

Two examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410,1990, respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information.This algorithm involves first identifying high scoring sequence pairs(HSPs) by identifying short words of length W in the query sequence,which either match or satisfy some positive-valued threshold score Twhen aligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are extendedin both directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a word length (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a word lengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, Proc.Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

The percent identity between two amino acid sequences can also bedetermined using the algorithm of E. Meyers and W. Miller, Comput. Appl.Biosci. 4:11-17, 1988) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4. In addition, the percent identitybetween two amino acid sequences can be determined using the Needlemanand Wunsch, J. Mol. Biol. 48:444-453, 1970) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix,and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1,2, 3, 4, 5, or 6.

Other than percentage of sequence identity noted above, anotherindication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

The term “nucleic acid” is used herein interchangeably with the term“polynucleotide” and refers to deoxyribonucleotides or ribonucleotidesand polymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, as detailed below,degenerate codon substitutions may be achieved by generating sequencesin which the third position of one or more selected (or all) codons issubstituted with mixed-base and/or deoxyinosine residues (Batzer et al.,(1991) Nucleic Acid Res. 19:5081; Ohtsuka et al., (1985) J. Biol. Chem.260:2605-2608; and Rossolini et al., (1994) Mol. Cell. Probes 8:91-98).

The term “operably linked” in the context of nucleic acids refers to afunctional relationship between two or more polynucleotide (e.g., DNA)segments. Typically, it refers to the functional relationship of atranscriptional regulatory sequence to a transcribed sequence. Forexample, a promoter or enhancer sequence is operably linked to a codingsequence if it stimulates or modulates the transcription of the codingsequence in an appropriate host cell or other expression system.Generally, promoter transcriptional regulatory sequences that areoperably linked to a transcribed sequence are physically contiguous tothe transcribed sequence, i.e., they are cis-acting. However, sometranscriptional regulatory sequences, such as enhancers, need not bephysically contiguous or located in close proximity to the codingsequences whose transcription they enhance.

The terms “polypeptide” and “protein” are used interchangeably herein torefer to a polymer of amino acid residues. The terms apply to amino acidpolymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymer. Unless otherwise indicated, a particularpolypeptide sequence also implicitly encompasses conservatively modifiedvariants thereof.

The term “immunoconjugate” or “antibody drug conjugate” as used hereinrefers to the linkage of an antibody or an antigen binding fragmentthereof with another agent, such as a chemotherapeutic agent, a toxin,an immunotherapeutic agent, an imaging probe, and the like. The linkagecan be covalent bonds, or non-covalent interactions such as throughelectrostatic forces. Various linkers, known in the art, can be employedin order to form the immunoconjugate. Additionally, the immunoconjugatecan be provided in the form of a fusion protein that may be expressedfrom a polynucleotide encoding the immunoconjugate. As used herein,“fusion protein” refers to proteins created through the joining of twoor more genes or gene fragments which originally coded for separateproteins (including peptides and polypeptides). Translation of thefusion gene results in a single protein with functional propertiesderived from each of the original proteins.

The term “subject” includes human and non-human animals. Non-humananimals include all vertebrates, e.g., mammals and non-mammals, such asnon-human primates, sheep, dog, cow, chickens, amphibians, and reptiles.Except when noted, the terms “patient” or “subject” are used hereininterchangeably.

The term “toxin,” “cytotoxin” or “cytotoxic agent” as used herein,refers to any agent that is detrimental to the growth and proliferationof cells and may act to reduce, inhibit, or destroy a cell ormalignancy.

The term “anti-cancer agent” as used herein refers to any agent that canbe used to treat a cell proliferative disorder such as cancer, includingbut not limited to, cytotoxic agents, chemotherapeutic agents,radiotherapy and radiotherapeutic agents, targeted anti-cancer agents,and immunotherapeutic agents.

The term “drug moiety” or “payload” as used herein refers to a chemicalmoiety that is conjugated to an antibody or antigen binding fragment,and can include any therapeutic or diagnostic agent, for example, ananti-cancer, anti-inflammatory, anti-infective (e.g., anti-fungal,antibacterial, anti-parasitic, anti-viral), or an anesthetic agent. Incertain aspects, a drug moiety is selected from a V-ATPase inhibitor, aHSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubulestabilizer, a microtubule destabilizer, an auristatin, a dolastatin, amaytansinoid, a MetAP (methionine aminopeptidase), an inhibitor ofnuclear export of proteins CRM1, a DPPIV inhibitor, an inhibitor ofphosphoryl transfer reactions in mitochondria, a protein synthesisinhibitor, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, aproteasome inhibitor, a kinesin inhibitor, an HDAC inhibitor, a DNAdamaging agent, a DNA alkylating agent, a DNA intercalator, a DNA minorgroove binder and a DHFR inhibitor. Methods for attaching each of theseto a linker compatible with the antibodies and method of the presentdisclosure are known in the art. See, e.g., Singh et al., (2009)Therapeutic Antibodies: Methods and Protocols, vol. 525, 445-457. Inaddition, a payload can be a biophysical probe, a fluorophore, a spinlabel, an infrared probe, an affinity probe, a chelator, a spectroscopicprobe, a radioactive probe, a lipid molecule, a polyethylene glycol, apolymer, a spin label, DNA, RNA, a protein, a peptide, a surface, anantibody, an antibody fragment, a nanoparticle, a quantum dot, aliposome, a PLGA particle, a saccharide or a polysaccharide.

The term “maytansinoid drug moiety” means the substructure of anantibody-drug conjugate that has the structure of a maytansinoidcompound. Maytansine was first isolated from the east African shrubMaytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and maytansinol analogues have been reported. SeeU.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814;4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946;4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866;4,424,219; 4,450,254; 4,362,663; and 4,371,533, and Kawai et al (1984)Chem. Pharm. Bull. 3441-3451), each of which are expressly incorporatedby reference. Specific examples of maytansinoids useful for conjugationinclude DM1, DM3 and DM4.

“Tumor” refers to neoplastic cell growth and proliferation, whethermalignant or benign, and all pre-cancerous and cancerous cells andtissues.

The term “anti-tumor activity” means a reduction in the rate of tumorcell proliferation, viability, or metastatic activity. A possible way ofshowing anti-tumor activity is to show a decline in growth rate of tumorcells, tumor size stasis or tumor size reduction. Such activity can beassessed using accepted in vitro or in vivo tumor models, including butnot limited to xenograft models, allograft models, MMTV models, andother known models known in the art to investigate anti-tumor activity.

The term “malignancy” refers to a non-benign tumor or a cancer. As usedherein, the term “cancer” includes a malignancy characterized byderegulated or uncontrolled cell growth. Exemplary cancers include:carcinomas, sarcomas, leukemias, and lymphomas.

The term “cancer” includes primary malignant tumors (e.g., those whosecells have not migrated to sites in the subject's body other than thesite of the original tumor) and secondary malignant tumors (e.g., thosearising from metastasis, the migration of tumor cells to secondary sitesthat are different from the site of the original tumor).

The term “cKIT” refers to a tyrosine kinase receptor that is a member ofthe receptor tyrosine kinase III family. The nucleic acid and amino acidsequences of cKIT are known, and have been published in GenBankAccession Nos. X06182.1, EU826594.1, GU983671.1, HM015525.1, HM015526.1,AK304031.1 and BC071593.1. See also SEQ ID NO:1 for the human cKIT cDNAsequence and SEQ ID NO.2 for the human cKIT protein sequence.Structurally, cKIT receptor is a type I transmembrane protein andcontains a signal peptide, 5 Ig-like C2 domains in the extracellulardomain and has a protein kinase domain in its intracellular domain andhas over its full length at least about 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% sequence identity with the amino acidsequence of SEQ ID NO.2. Structurally, a cKIT nucleic acid sequence hasover its full length at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity with the nucleic acid sequenceof SEQ ID NO 1.

The terms “cKIT expressing cancer” or “cKIT positive cancer” refers to acancer that express cKIT and/or a mutant form of cKIT on the surface ofcancer cells.

As used herein, the terms “treat,” “treating,” or “treatment” of anydisease or disorder refer in one aspect, to ameliorating the disease ordisorder (i.e., slowing or arresting or reducing the development of thedisease or at least one of the clinical symptoms thereof). In anotheraspect, “treat,” “treating,” or “treatment” refers to alleviating orameliorating at least one physical parameter including those which maynot be discernible by the patient. In yet another aspect, “treat,”“treating,” or “treatment” refers to modulating the disease or disorder,either physically, (e.g., stabilization of a discernible symptom),physiologically, (e.g., stabilization of a physical parameter), or both.In yet another aspect, “treat,” “treating,” or “treatment” refers topreventing or delaying the onset or development or progression of thedisease or disorder.

The term “therapeutically acceptable amount” or “therapeuticallyeffective dose” interchangeably refers to an amount sufficient to effectthe desired result (i.e., a reduction in tumor size, inhibition of tumorgrowth, prevention of metastasis, inhibition or prevention of viral,bacterial, fungal or parasitic infection). In some aspects, atherapeutically acceptable amount does not induce or cause undesirableside effects. A therapeutically acceptable amount can be determined byfirst administering a low dose, and then incrementally increasing thatdose until the desired effect is achieved. A “prophylactically effectivedosage,” and a “therapeutically effective dosage,” of the molecules ofthe present disclosure can prevent the onset of, or result in a decreasein severity of, respectively, disease symptoms, including symptomsassociated with cancer.

The term “co-administer” refers to the simultaneous presence of twoactive agents in the blood of an individual. Active agents that areco-administered can be concurrently or sequentially delivered.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows activity of c-Kit-MCC-DM1 ADCs in a subset of cancer celllines.

FIG. 2 depicts activity of 9P3-MCC-DM1, 9P3-SPDB-DM4 and 9P3-CX1-1-DM1in a subset of cancer cell lines.

FIG. 3 shows the activity of 9P3-MCC-DM1 in a panel of AML, GIST,melanoma and SCLC cell lines with varying levels of c-Kit surfacereceptor expression.

FIG. 4 shows the ability of c-Kit-MCC-DM1 ADCs to inhibit theproliferation of GIST-T1 (Imatinib-sensitive) cells.

FIG. 5 shows the ability of c-Kit-MCC-DM1 ADCs to inhibit theproliferation of GIST430 (Imatinib-resistant) cells.

FIG. 6 shows the ability of c-Kit-MCC-DM1 ADCs to inhibit theproliferation of NCI-H526 (higher c-Kit expressing SCLC) cells.

FIG. 7 shows the ability of c-Kit-MCC-DM1 ADCs to inhibit theproliferation of NCI-H1048 (lower c-Kit expressing SCLC) cells.

FIG. 8 shows the ability of c-Kit-MCC-DM1 ADCs to inhibit theproliferation of CMK11-5 (high c-Kit expressing AML) cells.

FIG. 9 shows the ability of c-Kit-MCC-DM1 ADCs to inhibit theproliferation of Uke-1 (lower c-Kit expressing AML) cells.

FIG. 10 is HDx-MS raw data plotted as the corrected difference over thestandard error in measurement. A more negative value indicates moreprotection from deuterium exchange upon binding of 9P3 to c-Kit antigen.The two most significant regions of protection are denoted as Region 1and Region 2.

FIG. 11 shows regions of HDx-MS protection are mapped using surfacefill: region 1 (black) and region 2 (dark grey). SCF binding sites aredenoted as Site I (light grey spheres), Site II (medium grey spheres),and Site III (darker grey spheres).

FIG. 12 is a Western blot showing the ability of SCF, NEG085-MCC-DM1,NEG024-MCC-DM1 and 20376-MCC-DM1 to modulate phosphorylation of c-Kit ina wildtype c-Kit cell line (Mo7e, A) or mutant c-Kit cell line (GIST-T1,B) after 15 minutes.

FIG. 13 shows that NEG085 and 20376 Abs mediate rapid internalization ofsurface c-Kit on GIST-T1 cells (A) and on human bone marrow cells (B)

FIG. 14 are Western blots showing the ability of SCF or NEG085-MCC-DM1to accelerate c-Kit degradation in a mutant c-Kit cell line (GIST-T1, A)and wildtype c-Kit cell line (NCI-H526, B) over a timecourse.

FIG. 15 shows the ability of NEG085, NEG024, 20376, NEG085-MCC-DM1 toinhibit the SCF-dependent proliferation of Mo7e cells.

FIG. 16 shows the ability of NEG085 and NEG085-MCC-DM1 to inhibitSCF-independent proliferation of Mo7e cells.

FIG. 17 shows the assessment of the ability of Campath (-CD52 Ab),NEG085 or 20376 antibodies to induce ADCC in vitro in Uke-1 cells.

FIG. 18 shows the assessment of the ability of NEG085 and 20376 tomediate primary human mast cell apoptosis.

FIG. 19 shows the assessment of the ability of NEG085 and 20376 tomediate primary human mast cell degranulation.

FIG. 20 shows co-localization of IgG1 and mitotic arrest ofNEG027-MCC-DM1 in GIST T1 xenograft model.

FIG. 21 shows tissue sections of mitotic arrest (p-histone H3) andapoptosis (caspase 3) after single dose of c-Kit ADC.

FIG. 22 graphically represents mitotic arrest and apoptosis induction 8days post single dose of c-Kit ADC.

FIG. 23 shows (A) Dose response efficacy in GIST T1 mouse xenograft and(B), change in body weight over course of treatment.

FIG. 24 graphically depicts (A) anti-DM1 ELISA after dosing in a GIST T1xenograft model and (B) anti-human IgG1 ELISA after dosing in a GIST T1xenograft model.

FIG. 25 is a table of NEG027-MCC-DM1 dose response in a GIST T1xenograft mouse model.

FIG. 26 are histology sections of NEG027-MCC-DM1 dose response efficacyin GIST T1. (A) is Group 4 pooled tumors, (B) Group 5 pooled tumors.

FIG. 27 depicts (A) Efficacy with 0.625 mg/kg in a GIST T1 xenograftmouse model, (B) change of tumor volume vs control (% T/C) and (C)change in body weight over course of treatment.

FIG. 28 shows clustering of day 41 after administration of single doseof anti-c-Kit ADC to a GIST T1 xenograft mouse.

FIG. 29 is a table of cKIT ADC efficacy at low effective dose in GIST T1xenograft model.

FIG. 30 shows (A) Anti-c-Kit PK in a GIST T1 xenograft mouse model,(left panel is anti-DM1 ELISA) (B) Right panel is anti-human IgG1 ELISA.

FIG. 31A-C shows (A) NEG085, NEG024 and NEG086 activity in a SCLC model(B) change in body weight over course of treatment (C) expression ofc-Kit on tumor sample.

FIG. 32 is a table of an anti-cKIT-ADC Efficacy Study in NCI-H1048 SCLC

FIG. 33 A-B shows (A) NEG085-MCC-DM1 dose response in NCI-H1048 (SCLC)xenograft model, (B) Change in body weight over course of treatment.

FIG. 34 is a table showing a NEG085-MCC-DM1 efficacy study in a NCI-1048(SCLC) xenograft mouse model.

FIG. 35 A-C shows (A) Efficacy of 20376 and NEG024 in NCI-H526 (SCLC)xenograft mouse model, (B) Antibody serum concentration after dosing and(C) IHC for c-Kit shows expression of cKIT levels on H526 tumor.

FIG. 36 shows anti-cKIT ADC in a small cell lung cancer (SCLC) xenograftmodel.

FIG. 37 shows anti-cKIT ADC efficacy in an AML xenograft model(Kasumi-1).

FIG. 38 shows anti-cKIT ADC efficacy in a HMC-1 mastocytosis xenograftmouse model.

FIG. 39 A/B shows efficacy of mouse cross reactive 20376-MCC-DM1 in GISTT1 xenograft mouse model with (A) dosage and tumor volume and (B) changein body weight over course of treatment.

FIG. 40 A/B shows (A) Efficacy of mouse cross reactive 20376-MCC-DM1 inGIST T1 xenograft mouse model -PK and (B) Antibody serum concentrationpost dosing.

FIG. 41 shows dose response efficacy study in GIST T1 SCID-beige mice.

FIG. 42 A/B shows (A) efficacy in GIST T1 xenograft mouse model (noefficacy with unconjugated) and (B) change in body weight over course oftreatment.

FIG. 43 is a comparison of efficacy in a GIST T1 mouse xenograft model(unlabeled/MCC-DM1/SPDB-DM4).

FIG. 44 A/B shows (A)Efficacy in a GIST 430 xenograft model comparingSPDB-DM4 and MCC-DM1 and (B) Change in body weight over course oftreatment

FIG. 45 shows efficacy in GIST 430 SCID-beige mouse model.

DETAILED DESCRIPTION

The present disclosure provides for antibodies, antibody fragments(e.g., antigen binding fragments), and antibody drug conjugates thatbind to cKIT. In particular, the present disclosure is directed toantibodies and antibody fragments (e.g., antigen binding fragments) thatbind to cKIT, and internalize upon such binding. The antibodies andantibody fragments (e.g., antigen binding fragments) of the presentdisclosure can be used for producing antibody drug conjugates.Furthermore, the present disclosure provides antibody drug conjugatesthat have desirable pharmacokinetic characteristics and other desirableattributes, and thus can be used for treating cancer expressing cKIT,without limitation, for example: gastrointestinal stromal tumors (GIST),small cell lung cancer (SCLC), acute myelod leukemia (AML), melanoma,mast cell leukemia (MCL), mastocytosis, neurofibromatosis, breastcancer, non-small cell lung cancer (NSCLC) and pancreatic cancer. Thepresent disclosure further provides pharmaceutical compositionscomprising the antibody drug conjugates, and methods of making and usingsuch pharmaceutical compositions for the treatment of cancer.

Antibody Drug Conjugates

The present disclosure provides antibody drug conjugates, where anantibody, antigen binding fragment or its functional equivalent thatspecifically binds to cKIT is linked to a drug moiety. In one aspect,the antibodies, antigen binding fragments or their functionalequivalents are linked, via covalent attachment by a linker, to a drugmoiety that is an anti-cancer agent. The antibody drug conjugates canselectively deliver an effective dose of an anti-cancer agent (e.g., acytotoxic agent) to tumor tissues expressing cKIT, whereby greaterselectivity (and lower efficacious dose) may be achieved.

In one aspect, the disclosure provides for an immunoconjugate of Formula(I):

Ab-(L-(D)_(m))_(n)

Wherein Ab represents an cKIT binding antibody or antibody fragment(e.g., antigen binding fragment) described herein;L is a linker;D is a drug moiety;m is an integer from 1-8; andn is an integer from 1-20. In one aspect, n is an integer from 1 to 10,2 to 8, or 2 to 5. In a specific aspect, n is 3 to 4. In some aspects, mis 1. In some aspects, m is 2, 3 or 4.

While the drug to antibody ratio has an exact value for a specificconjugate molecule (e.g., n multiplied by m in Formula (I)), it isunderstood that the value will often be an average value when used todescribe a sample containing many molecules, due to some degree ofinhomogeneity, typically associated with the conjugation step. Theaverage loading for a sample of an immunoconjugate is referred to hereinas the drug to antibody ratio, or “DAR.” In the aspect of maytansinoids,this can be referred to as maytansinoid to antibody ratio or “MAR.” Insome aspects, the DAR is between about 1 and about 5, and typically isabout 3, 3.5, 4, 4.5, or 5. In some aspects, at least 50% of a sample byweight is compound having the average DAR plus or minus 2, andpreferably at least 50% of the sample is a conjugate that contains theaverage DAR plus or minus 1. Other aspects include immunoconjugateswherein the DAR is about 3.5. In some aspects, a DAR of ‘about n’ meansthe measured value for DAR is within 20% of n.

The present disclosure provides immunoconjugates comprising theantibodies, antibody fragments (e.g., antigen binding fragments) andtheir functional equivalents as disclosed herein, linked or conjugatedto a drug moiety. In one aspect, the drug moiety D is a maytansinoiddrug moiety, including those having the structure:

where the wavy line indicates the covalent attachment of the sulfur atomof the maytansinoid to a linker of an antibody drug conjugate. R at eachoccurrence is independently H or a C₁-C₆ alkyl. The alkylene chainattaching the amide group to the sulfur atom may be methanyl, ethanyl,or proyl, i.e. m is 1, 2, or 3. (U.S. Pat. No. 633,410, U.S. Pat. No.5,208,020, Chari et al. (1992) Cancer Res. 52; 127-131, Lui et al.(1996) Proc. Natl. Acad. Sci. 93:8618-8623).

All stereoisomers of the maytansinoid drug moiety are contemplated forthe immunoconjugates disclosed, i.e. any combination of R and Sconfigurations at the chiral carbons of the maytansinoid. In one aspectthe maytansinoid drug moiety has the following stereochemistry.

In one aspect, the maytansinoid drug moiety isN^(2′)-deacetyl-N^(2′)-(3-mercapto-1-oxopropyl)-maytansine (also knownas DM1). DM1 is represented by the following structural formula.

In another aspect the maytansinoid drug moiety is N^(2′-deacetyl-N)^(2′)-(4-mercapto-1-oxopentyl)-maytansine (also known as DM3). DM3 isrepresented by the following structural formula.

In another aspect the maytansinoid drug moiety isN^(2′-deacetyl-N)2′-(4-methyl-4-mercapto-1-oxopentyl)-maytansine (alsoknown as DM4). DM4 is represented by the following structural formula.

The drug moiety D can be linked to the antibody through a linker L. L isany chemical moiety that is capable of linking the antibody Ab to thedrug moiety D. The linker, L attaches the antibody Ab to the drug Dthrough covalents bond(s). The linker reagent is a bifunctional ormultifunctional moiety which can be used to link a drug moiety D and anantibody Ab to form antibody drug conjugates. Antibody drug conjugatescan be prepared using a linker having a reactive functionality forbinding to the drug moiety D and to the antibody Ab. A cysteine, thiolor an amine, e.g. N-terminus or amino acid side chain such as lysine ofthe antibody can form a bond with a functional group of a linkerreagent.

In one aspect, L is a cleavable linker. In another aspect, L is anon-cleavable linker. In some aspects, L is an acid-labile linker,photo-labile linker, peptidase cleavable linker, esterase cleavablelinker, a disulfide bond reducible linker, a hydrophilic linker, aprocharged linker, or a dicarboxylic acid based linker.

Suitable cross-linking reagents that form a non-cleavable linker betweenthe drug moiety D, for example maytansinoid, and the antibody Ab arewell known in the art, and can form non-cleavable linkers that comprisea sulfur atom (such as SMCC) or those that are without a sulfur atom.Preferred cross-linking reagents that form non-cleavable linkers betweenthe drug moiety D, for example maytansinoid, and the antibody Abcomprise a maleimido- or haloacetyl-based moiety. According to thepresent disclosure, such non-cleavable linkers are said to be derivedfrom maleimido- or haloacetyl-based moieties.

Cross-linking reagents comprising a maleimido-based moiety include butnot limited to, N-succinimidyl-4-(maleimidomethyl)cyclohexanecarboxylate(SMCC), sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-SMCC),N-succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate),which is a “long chain” analog of SMCC (LC-SMCC), K-maleimidoundeconoicacid N-succinimidyl ester (KMUA), γ-maleimidobutyric acid N-succinimidylester (GMBS), ε-maleimidocaproic acid N-succinimidyl ester (EMCS),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),N-(α-maleimidoacetoxy)-succinimide ester (AMSA),succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH),N-succinimidyl-4-(p-maleimidophenyl)-butyrate (SMPB),N-(-p-maleomidophenyl)isocyanate (PMIP) and maleimido-basedcross-linking reagents containing a polyethylene glycol spacer, such asMAL-PEG-NHS. These cross-linking reagents form non-cleavable linkersderived from maleimido-based moieties. Representative structures ofmaleimido-based cross-linking reagents are shown below.

In another aspect, the linker L is derived fromN-succinimidyl-4-(maleimidomethyl)cyclohexanecarboxylate (SMCC),sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-SMCC) or MAL-PEG-NHS.

Cross-linking reagents comprising a haloacetyle-based moiety includeN-succinimidyl iodoacetate (SIA),N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), N-succinimidylbromoacetate (SBA) and N-succinimidyl 3-(bromoacetamido)propionate(SBAP). These cross-linking reagents form a non-cleavable linker derivedfrom haloacetyl-based moieties. Representative structures ofhaloacetyl-based cross-linking reagents are shown below.

In one aspect, the linker L is derived from N-succinimidyl iodoacetate(SIA) or N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB).

Suitable cross-linking reagents that form a cleavable linker between thedrug moiety D, for example maytansinoid, and the antibody Ab are wellknown in the art. Disulfide containing linkers are linkers cleavablethrough disulfide exchange, which can occur under physiologicalconditions. According to the present disclosure, such cleavable linkersare said to be derived from disulfide-based moieties. Suitable disulfidecross-linking reagents includeN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP),N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB) andN-succinimidyl-4-(2-pyridyldithio)-2-sulfo-butanoate (sulfo-SPDB), thestructures of which are shown below. These disulfide cross-linkingreagents form a cleavable linker derived from disulfide-based moieties.

In one aspect, the linker L is derived fromN-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB).

Suitable cross-linking reagents that form a charged linker between thedrug moiety D, for example maytansinoid, and the antibody Ab are knownas procharged cross-linking reagents. In one aspect, the linker L isderived from the procharged cross-linking reagent is CX1-1. Thestructure of CX1-1 is below.

In one aspect provided by the disclosure, the conjugate is representedby any one of the following structural formulae:

wherein:

Ab is an antibody or antigen binding fragment thereof that specificallybinds to human cKIT;

n, which indicates the number of D-L groups attached the Ab through theformation of an amide bond with a primary amine of the Ab, is an integerfrom 1 to 20. In one aspect, n is an integer from 1 to 10, 2 to 8 or 2to 5. In a specific aspect, n is 3 or 4.

In one aspect, the average molar ratio of drug (e.g., DM1 or DM4) to theantibody in the conjugate (i.e., average w value, also known asMaytanisnoid Antibody Ratio (MAR)) is about 1 to about 10, about 2 toabout 8 (e.g., 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3,4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1,7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, or 8.1), about 2.5 to about7, about 3 to about 5, about 2.5 to about 4.5 (e.g., about 2.5, about2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.3,about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5), about 3.0to about 4.0, about 3.2 to about 4.2, or about 4.5 to 5.5 (e.g., about4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1,about 5.2, about 5.3, about 5.4, or about 5.5).

In one aspect provided by the disclosure, the conjugate hassubstantially high purity and has one or more of the following features:(a) greater than about 90% (e.g., greater than or equal to about 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%), preferably greaterthan about 95%, of conjugate species are monomeric, (b) unconjugatedlinker level in the conjugate preparation is less than about 10% (e.g.,less than or equal to about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%)(relative to total linker), (c) less than 10% of conjugate species arecrosslinked (e.g., less than or equal to about 9%, 8%, 7%, 6%, 5%, 4%,3%, 2%, 1%, or 0%), (d) free drug (e.g., DM1 or DM4) level in theconjugate preparation is less than about 2% (e.g., less than or equal toabout 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%,0.4%, 0.3%, 0.2%, 0.1%, or 0%) (mol/mol relative to total cytotoxicagent).

As used herein, the term “unconjugated linker” refers to the antibodythat is covalently linked with a linker derived from a cross-linkingreagent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1), wherein theantibody is not covalently coupled to the drug (e.g., DM1 or DM4)through a linker (i.e., the “unconjugated linker” can be represented byAb-SMCC, Ab-SPDB, or Ab-CX1-1).

1. Drug Moiety

The present disclosure provides immunoconjugates that specifically bindto cKIT. The immunoconjugates of the present disclosure compriseanti-cKIT antibodies, antibody fragments (e.g., antigen bindingfragments) or functional equivalents that are conjugated to a drugmoiety, e.g., an anti-cancer agent, anti-hematological disorder agent,an autoimmune treatment agent, an anti-inflammatory agent, an antifungalagent, an antibacterial agent, an anti-parasitic agent, an anti-viralagent, or an anesthetic agent. The antibodies, antibody fragments (e.g.,antigen binding fragments) or functional equivalents can be conjugatedto several identical or different drug moieties using any methods knownin the art.

In certain aspects, the drug moiety of the immunoconjugates of thepresent disclosure is selected from a group consisting of a V-ATPaseinhibitor, a pro-apoptotic agent, a Bcl2 inhibitor, an MCL1 inhibitor, aHSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubulestabilizer, a microtubule destabilizer, an auristatin, a dolastatin, amaytansinoid, a MetAP (methionine aminopeptidase), an inhibitor ofnuclear export of proteins CRM1, a DPPIV inhibitor, proteasomeinhibitors, an inhibitor of phosphoryl transfer reactions inmitochondria, a protein synthesis inhibitor, a kinase inhibitor, a CDK2inhibitor, a CDK9 inhibitor, a kinesin inhibitor, an HDAC inhibitor, aDNA damaging agent, a DNA alkylating agent, a DNA intercalator, a DNAminor groove binder and a DHFR inhibitor.

In one aspect, the drug moiety of the immunoconjugates of the presentdisclosure is a maytansinoid drug moiety, such as but not limited to,DM1, DM3, or DM4.

Further, the antibodies, antibody fragments (e.g., antigen bindingfragments) or functional equivalents of the present disclosure may beconjugated to a drug moiety that modifies a given biological response.Drug moieties are not to be construed as limited to classical chemicaltherapeutic agents. For example, the drug moiety may be a protein,peptide, or polypeptide possessing a desired biological activity. Suchproteins may include, for example, a toxin such as abrin, ricin A,pseudomonas exotoxin, cholera toxin, or diphtheria toxin, a protein suchas tumor necrosis factor, α-interferon, β-interferon, nerve growthfactor, platelet derived growth factor, tissue plasminogen activator, acytokine, an apoptotic agent, an anti-angiogenic agent, or, a biologicalresponse modifier such as, for example, a lymphokine.

In one aspect, the antibodies, antibody fragments (e.g., antigen bindingfragments) or functional equivalents of the present disclosure areconjugated to a drug moiety, such as a cytotoxin, a drug (e.g., animmunosuppressant) or a radiotoxin. Examples of cytotoxin include butare not limited to, taxanes (see, e.g., International (PCT) PatentApplication Nos. WO 01/38318 and PCT/US03/02675), DNA-alkylating agents(e.g., CC-1065 analogs), anthracyclines, tubulysin analogs, duocarmycinanalogs, auristatin E, auristatin F, maytansinoids, and cytotoxic agentscomprising a reactive polyethylene glycol moiety (see, e.g., Sasse etal., J. Antibiot. (Tokyo), 53, 879-85 (2000), Suzawa et al., Bioorg.Med. Chem., 8, 2175-84 (2000), Ichimura et al., J. Antibiot. (Tokyo),44, 1045-53 (1991), Francisco et al., Blood (2003) (electronicpublication prior to print publication), U.S. Pat. Nos. 5,475,092,6,340,701, 6,372,738, and 6,436,931, U.S. Patent Application PublicationNo. 2001/0036923 A1, Pending U.S. patent application Ser. Nos.10/024,290 and 10/116,053, and International (PCT) Patent ApplicationNo. WO 01/49698), taxon, cytochalasin B, gramicidin D, ethidium bromide,emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, t.colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione,mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. Therapeutic agents alsoinclude, for example, anti-metabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), ablating agents (e.g., mechlorethamine, thiotepachlorambucil, meiphalan, carmustine (BSNU) and lomustine (CCNU),cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin, anthracyclines(e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics(e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, andanthramycin (AMC)), and anti-mitotic agents (e.g., vincristine andvinblastine). (See e.g., Seattle Genetics US20090304721).

Other examples of cytotoxins that can be conjugated to the antibodies,antibody fragments (antigen binding fragments) or functional equivalentsof the present disclosure include duocarmycins, calicheamicins,maytansines and auristatins, and derivatives thereof.

Various types of cytotoxins, linkers and methods for conjugatingtherapeutic agents to antibodies are known in the art, see, e.g., Saitoet al., (2003) Adv. Drug Deliv. Rev. 55:199-215; Trail et al., (2003)Cancer Immunol. Immunother. 52:328-337; Payne, (2003) Cancer Cell3:207-212; Allen, (2002) Nat. Rev. Cancer 2:750-763; Pastan andKreitman, (2002) Curr. Opin. Investig. Drugs 3:1089-1091; Senter andSpringer, (2001) Adv. Drug Deliv. Rev. 53:247-264.

The antibodies, antibody fragments (e.g., antigen binding fragments) orfunctional equivalents of the present disclosure can also be conjugatedto a radioactive isotope to generate cytotoxic radiopharmaceuticals,referred to as radioimmunoconjugates. Examples of radioactive isotopesthat can be conjugated to antibodies for use diagnostically ortherapeutically include, but are not limited to, iodine-131, indium-111,yttrium-90, and lutetium-177. Methods for preparingradioimmunoconjugates are established in the art. Examples ofradioimmunoconjugates are commercially available, including Zevalin™(DEC Pharmaceuticals) and Bexxar™ (Corixa Pharmaceuticals), and similarmethods can be used to prepare radioimmunoconjugates using theantibodies disclosed herein. In certain aspects, the macrocyclicchelator is 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid(DOTA) which can be attached to the antibody via a linker molecule. Suchlinker molecules are commonly known in the art and described in Denardoet al., (1998) Clin Cancer Res. 4(10):2483-90; Peterson et al., (1999)Bioconjug. Chem. 10(4):553-7; and Zimmerman et al., (1999) Nucl. Med.Biol. 26(8):943-50, each incorporated by reference in their entireties.

The antibodies, antibody fragments (e.g., antigen binding fragments) orfunctional equivalents of the present disclosure can also conjugated toa heterologous protein or polypeptide (or fragment thereof, preferablyto a polypeptide of at least 10, at least 20, at least 30, at least 40,at least 50, at least 60, at least 70, at least 80, at least 90 or atleast 100 amino acids) to generate fusion proteins. In particular, thepresent disclosure provides fusion proteins comprising an antibodyfragment (e.g., antigen binding fragment) described herein (e.g., a Fabfragment, Fd fragment, Fv fragment, F(ab)₂ fragment, a VH domain, a VHCDR, a VL domain or a VL CDR) and a heterologous protein, polypeptide,or peptide.

Additional fusion proteins may be generated through the techniques ofgene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling(collectively referred to as “DNA shuffling”). DNA shuffling may beemployed to alter the activities of antibodies of the present disclosureor fragments thereof (e.g., antibodies or fragments thereof with higheraffinities and lower dissociation rates). See, generally, U.S. Pat. Nos.5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten etal., (1997) Curr. Opinion Biotechnol. 8:724-33; Harayama, (1998) TrendsBiotechnol. 16(2):76-82; Hansson et al., (1999) J. Mol. Biol.287:265-76; and Lorenzo and Blasco, (1998) Biotechniques 24(2):308-313(each of these patents and publications are hereby incorporated byreference in its entirety). Antibodies or fragments thereof, or theencoded antibodies or fragments thereof, may be altered by beingsubjected to random mutagenesis by error-prone PCR, random nucleotideinsertion or other methods prior to recombination. A polynucleotideencoding an antibody or fragment thereof that specifically binds to anantigen may be recombined with one or more components, motifs, sections,parts, domains, fragments, etc. of one or more heterologous molecules.

Moreover, the antibodies, antibody fragments (e.g., antigen bindingfragments) or functional equivalents of the present disclosure can beconjugated to marker sequences, such as a peptide, to facilitatepurification. In preferred aspects, the marker amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., (1989) Proc. Natl. Acad. Sci. USA 86:821-824, for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the hemagglutinin (“HA”) tag, which corresponds to anepitope derived from the influenza hemagglutinin protein (Wilson et al.,(1984) Cell 37:767), and the “FLAG” tag (A. Einhauer et al., J. Biochem.Biophys. Methods 49: 455-465, 2001). As described in the presentdisclosure, antibodies or antigen binding fragments can also beconjugated to tumor-penetrating peptides in order to enhance theirefficacy.

In other aspects, the antibodies, antibody fragments (e.g., antigenbinding fragments) or functional equivalents of the present disclosureare conjugated to a diagnostic or detectable agent. Suchimmunoconjugates can be useful for monitoring or prognosing the onset,development, progression and/or severity of a disease or disorder aspart of a clinical testing procedure, such as determining the efficacyof a particular therapy. Such diagnosis and detection can beaccomplished by coupling the antibody to detectable substancesincluding, but not limited to, various enzymes, such as, but not limitedto, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; prosthetic groups, such as, but not limited to,streptavidin/biotin and avidin/biotin; fluorescent materials, such as,but not limited to, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430,Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532,Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594,Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660,Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, umbelliferone,fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;luminescent materials, such as, but not limited to, luminol;bioluminescent materials, such as but not limited to, luciferase,luciferin, and aequorin; radioactive materials, such as, but not limitedto, iodine (¹³¹I, ¹²⁵I, ¹²³I, and ¹²¹ I) carbon (¹⁴C), sulfur (³⁵S),tritium (³H), indium (¹¹⁵In, ¹¹³In, ¹¹²In, and ¹¹¹In,), technetium(⁹⁹Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd),molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd,¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru,⁶⁸Ge, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ⁶⁴Cu, ¹¹³Sn, and ¹¹⁷Sn; andpositron emitting metals using various positron emission tomographies,and non-radioactive paramagnetic metal ions.

The antibodies, antibody fragments (e.g., antigen binding fragments) orfunctional equivalents of the present disclosure may also be attached tosolid supports, which are particularly useful for immunoassays orpurification of the target antigen. Such solid supports include, but arenot limited to, glass, cellulose, polyacrylamide, nylon, polystyrene,polyvinyl chloride or polypropylene.

2. Linker

As used herein, a “linker” is any chemical moiety that is capable oflinking an antibody, antibody fragment (e.g., antigen binding fragments)or functional equivalent to another moiety, such as a drug moeity.Linkers can be susceptible to cleavage (cleavable linker), such as,acid-induced cleavage, photo-induced cleavage, peptidase-inducedcleavage, esterase-induced cleavage, and disulfide bond cleavage, atconditions under which the compound or the antibody remains active.Alternatively, linkers can be substantially resistant to cleavage (e.g.,stable linker or noncleavable linker). In some aspects, the linker is aprocharged linker, a hydrophilic linker, or a dicarboxylic acid basedlinker.

In one aspect, the linker used is derived from a crosslinking reagentsuch as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl4-(2-pyridyldithio)butanoate (SPDB),sulfosuccinimidyl-4-(2-pyridyldithio)butanoate (sulfo-SPDB),N-succinimidyl iodoacetate (SIA),N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), maleimide PEG NHS,N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC),N-sulfosuccinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate(sulfo-SMCC) or 2,5-dioxopyrrolidin-1-yl17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazaheptadecan-1-oate(CX1-1). In another aspect, the linker used is derived from across-linking agent such as N-succinimidyl-3-(2-pyridyldithio)propionate(SPDP), N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC),N-sulfosuccinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate(sulfo-SMCC), sulfosuccinimidyl-4-(2-pyridyldithio)butanoate(sulfo-SPDB) or 2,5-dioxopyrrolidin-1-yl17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazaheptadecan-1-oate(CX1-1).

Non-cleavable linkers are any chemical moiety capable of linking a drug,such as a maytansinoid, to an antibody in a stable, covalent manner anddoes not fall off under the categories listed above for cleaveablelinkers. Thus, non-cleavable linkers are substantialy resistant toacid-induced cleavage, photo-induced cleavage, peptidase-inducedcleavage, esterase-induced cleavage and disulfide bond cleavage.Furthermore, non-cleavable refers to the ability of the chemical bond inthe linker or adjoining to the linker to withstand cleavage induced byan acid, photolabile-cleaving agent, a peptidase, an esterase, or achemical or physiological compound that cleaves a disulfide bond, atconditions under which the drug, such as maytansionoid or the antibodydoes not lose its activity.

Acid-labile linkers are linkers cleavable at acidic pH. For example,certain intracellular compartments, such as endosomes and lysosomes,have an acidic pH (pH 4-5), and provide conditions suitable to cleaveacid-labile linkers.

Photo-labile linkers are linkers that are useful at the body surface andin many body cavities that are accessible to light. Furthermore,infrared light can penetrate tissue.

Some linkers can be cleaved by peptidases, i.e. peptidase cleavablelinkers. Only certain peptides are readily cleaved inside or outsidecells, see e.g. Trout et al., 79 Proc. Natl. Acad. Sci. USA, 626-629(1982) and Umemoto et al. 43 Int. J. Cancer, 677-684 (1989).Furthermore, peptides are composed of α-amino acids and peptidic bonds,which chemically are amide bonds between the carboxylate of one aminoacid and the amino group of a second amino acid. Other amide bonds, suchas the bond between a carboxylate and the ε-amino group of lysine, areunderstood not to be peptidic bonds and are considered non-cleavable.

Some linkers can be cleaved by esterases, i.e. esterase cleavablelinkers. Again, only certain esters can be cleaved by esterases presentinside or outside of cells. Esters are formed by the condensation of acarboxylic acid and an alcohol. Simple esters are esters produced withsimple alcohols, such as aliphatic alcohols, and small cyclic and smallaromatic alcohols.

Procharged linkers are derived from charged cross-linking reagents thatretain their charge after incorporation into an antibody drug conjugate.Examples of procharged linkers can be found in US 2009/0274713.

3. Conjugation and Preparation of ADCs

The conjugates of the present disclosure can be prepared by any methodsknown in the art, such as those described in U.S. Pat. Nos. 7,811,572,6,411,163, 7,368,565, and 8,163,888, and US application publications2011/0003969, 2011/0166319, 2012/0253021 and 2012/0259100. The entireteachings of these patents and patent application publications areherein incorporated by reference.

One-Step Process

In one aspect, the conjugates of the present disclosure can be preparedby a one-step process. The process comprises combining the antibody,drug and cross-linking agent in a substantially aqueous medium,optionally containing one or more co-solvents, at a suitable pH. In oneaspect, the process comprises the step of contacting the antibody of thepresent disclosure with a drug (e.g., DM1 or DM4) to form a firstmixture comprising the antibody and the drug, and then contacting thefirst mixture comprising the antibody and the drug with a cross-linkingagent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1) in a solutionhaving a pH of about 4 to about 9 to provide a mixture comprising (i)the conjugate (e.g., Ab-SMCC-DM1, Ab-SPDB-DM4, or Ab-CX1-1-DM1), (ii)free drug (e.g., DM1 or DM4), and (iii) reaction by-products.

In one aspect, the one-step process comprises contacting the antibodywith the drug (e.g., DM1 or DM4) and then the cross-linking agent (e.g.,SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1) in a solution having a pHof about 6 or greater (e.g., about 6 to about 9, about 6 to about 7,about 7 to about 9, about 7 to about 8.5, about 7.5 to about 8.5, about7.5 to about 8.0, about 8.0 to about 9.0, or about 8.5 to about 9.0).For example, the process comprises contacting a cell-binding agent withthe drug (DM1 or DM4) and then the cross-linking agent (e.g., SMCC,Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1) in a solution having a pH ofabout 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2,about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5,about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0. In anotheraspect, the process comprises contacting a cell-binding agent with thedrug (e.g., DM1 or DM4) and then the cross-linking agent (e.g., SMCC,Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1) in a solution having a pH ofabout 7.8 (e.g., a pH of 7.6 to 8.0 or a pH of 7.7 to 7.9).

The one-step process (i.e., contacting the antibody with the drug (e.g.,DM1 or DM4) and then the cross-linking agent (e.g., SMCC, Sulfo-SMCC,SPDB, Sulfo-SPDB or CX1-1) can be carried out at any suitabletemperature known in the art. For example, the one-step process canoccur at about 20° C. or less (e.g., about −10° C. (provided that thesolution is prevented from freezing, e.g., by the presence of organicsolvent used to dissolve the cytotoxic agent and the bifunctionalcrosslinking reagent) to about 20° C., about 0° C. to about 18° C.,about 4° C. to about 16° C.), at room temperature (e.g., about 20° C. toabout 30° C. or about 20° C. to about 25° C.), or at an elevatedtemperature (e.g., about 30° C. to about 37° C.). In one aspect, theone-step process occurs at a temperature of about 16° C. to about 24° C.(e.g., about 16° C., about 17° C., about 18° C., about 19° C., about 20°C., about 21° C., about 22° C., about 23° C., about 24° C., or about 25°C.). In another aspect, the one-step process is carried out at atemperature of about 15° C. or less (e.g., about −10° C. to about 15°C., or about 0° C. to about 15° C.). For example, the process comprisescontacting the antibody with the drug (e.g., DM1 or DM4) and then thecross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1)at a temperature of about 15° C., about 14° C., about 13° C., about 12°C., about 11° C., about 10° C., about 9° C., about 8° C., about 7° C.,about 6° C., about 5° C., about 4° C., about 3° C., about 2° C., about1° C., about 0° C., about −1° C., about −2° C., about −3° C., about −4°C., about −5° C., about −6° C., about −7° C., about −8° C., about −9°C., or about −10° C., provided that the solution is prevented fromfreezing, e.g., by the presence of organic solvent(s) used to dissolvethe cross-linking agent (e.g., SMCC, Sulfo-SMCC, Sulfo-SPDB SPDB, orCX1-1). In one aspect, the process comprises contacting the antibodywith the drug (e.g., DM1 or DM4) and then the cross-linking agent (e.g.,SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1) at a temperature of about−10° C. to about 15° C., about 0° C. to about 15° C., about 0° C. toabout 10° C., about 0° C. to about 5° C., about 5° C. to about 15° C.,about 10° C. to about 15° C., or about 5° C. to about 10° C. In anotheraspect, the process comprises contacting the antibody with the drug(e.g., DM1 or DM4) and then the cross-linking agent (e.g., SMCC,Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1) at a temperature of about 10° C.(e.g., a temperature of 8° C. to 12° C. or a temperature of 9° C. to 11°C.).

In one aspect, the contacting described above is effected by providingthe antibody, then contacting the antibody with the drug (e.g., DM1 orDM4) to form a first mixture comprising the antibody and the drug (e.g.,DM1 or DM4), and then contacting the first mixture comprising theantibody and the drug (e.g., DM1 or DM4) with the cross-linking agent(e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1). For example, in oneaspect, the antibody is provided in a reaction vessel, the drug (e.g.,DM1 or DM4) is added to the reaction vessel (thereby contacting theantibody), and then the cross-linking agent (e.g., SMCC, Sulfo-SMCC,SPDB, Sulfo-SPDB or CX1-1) is added to the mixture comprising theantibody and the drug (e.g., DM1 or DM4) (thereby contacting the mixturecomprising the antibody and the drug). In one aspect, the antibody isprovided in a reaction vessel, and the drug (e.g., DM1 or DM4) is addedto the reaction vessel immediately following providing the antibody tothe vessel. In another aspect, the antibody is provided in a reactionvessel, and the drug (e.g., DM1 or DM4) is added to the reaction vesselafter a time interval following providing the antibody to the vessel(e.g., about 5 minutes, about 10 minutes, about 20 minutes, about 30minutes, about 40 minutes, about 50 minutes, about 1 hour, about 1 dayor longer after providing the cell-binding agent to the space). The drug(e.g., DM1 or DM4) can be added quickly (i.e., within a short timeinterval, such as about 5 minutes, about 10 minutes) or slowly (such asby using a pump).

The mixture comprising the antibody and the drug (e.g., DM1 or DM4) canthen be contacted with the cross-linking agent (e.g., SMCC, Sulfo-SMCC,SPDB, Sulfo-SPDB or CX1-1) either immediately after contacting theantibody with the drug (e.g., DM1 or DM4) or at some later point (e.g.,about 5 minutes to about 8 hours or longer) after contacting theantibody with the drug (e.g., DM1 or DM4). For example, in one aspect,the cross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB orCX1-1) is added to the mixture comprising the antibody and the drug(e.g., DM1 or DM4) immediately after the addition of the drug (e.g., DM1or DM4) to the reaction vessel comprising the antibody. Alternatively,the mixture comprising the antibody and the drug (e.g., DM1 or DM4) canbe contacted with the cross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB,Sulfo-SPDB or CX1-1) at about 5 minutes, about 10 minutes, about 20minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours,about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8hours, or longer after contacting the antibody with the drug (e.g., DM1or DM4).

After the mixture comprising the antibody and the drug (e.g., DM1 orDM4) is contacted with the cross-linking agent (e.g., SMCC, Sulfo-SMCC,SPDB, Sulfo-SPDB or CX1-1) the reaction is allowed to proceed for about1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours,about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours,about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours,about 24 hours, or longer (e.g., about 30 hours, about 35 hours, about40 hours, about 45 hours, or about 48 hrs).

In one aspect, the one-step process further comprises a quenching stepto quench any unreacted drug (e.g., DM1 or DM4) and/or unreactedcross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1).The quenching step is typically performed prior to purification of theconjugate. In one aspect, the mixture is quenched by contacting themixture with a quenching reagent. As used herein, the “quenchingreagent” refers to a reagent that reacts with the free drug (e.g., DM1or DM4) and/or cross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB,Sulfo-SPDB or CX1-1). In one aspect, maleimide or haloacetamidequenching reagents, such as 4-maleimidobutyric acid,3-maleimidopropionic acid, N-ethylmaleimide, iodoacetamide, oriodoacetamidopropionic acid, can be used to ensure that any unreactedgroup (such as thiol) in the drug (e.g., DM1 or DM4) is quenched. Thequenching step can help prevent the dimerization of the drug (e.g.,DM1). The dimerized DM1 can be difficult to remove. Upon quenching withpolar, charged thiol-quenching reagents (such as 4-maleimidobutyric acidor 3-maleimidopropionic acid), the excess, unreacted DM1 is convertedinto a polar, charged, water-soluble adduct that can be easily separatedfrom the covalently-linked conjugate during the purification step.Quenching with non-polar and neutral thiol-quenching reagents can alsobe used. In one aspect, the mixture is quenched by contacting themixture with a quenching reagent that reacts with the unreactedcross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1).For example, nucleophiles can be added to the mixture in order to quenchany unreacted SMCC. The nucleophile preferably is an amino groupcontaining nucleophile, such as lysine, taurine and hydroxylamine.

In another aspect, the reaction (i.e., contacting the antibody with thedrug (e.g., DM1 or DM4) and then cross-linking agent (e.g., SMCC,Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1)) is allowed to proceed tocompletion prior to contacting the mixture with a quenching reagent. Inthis regard, the quenching reagent is added to the mixture about 1 hourto about 48 hours (e.g., about 1 hour, about 2 hours, about 3 hours,about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours,about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours,about 22 hours, about 23 hours, about 24 hours, or about 25 hours toabout 48 hours) after the mixture comprising the antibody and the drug(e.g., DM1 or DM4) is contacted with the cross-linking agent (e.g.,SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1).

Alternatively, the mixture is quenched by lowering the pH of the mixtureto about 5.0 (e.g., 4.8, 4.9, 5.0, 5.1 or 5.2). In another aspect, themixture is quenched by lowering the pH to less than 6.0, less than 5.5,less than 5.0, less than 4.8, less than 4.6, less than 4.4, less than4.2, less than 4.0. Alternatively, the pH is lowered to about 4.0 (e.g.,3.8, 3.9, 4.0, 4.1 or 4.2) to about 6.0 (e.g., 5.8, 5.9, 6.0, 6.1 or6.2), about 4.0 to about 5.0, about 4.5 (e.g., 4.3, 4.4, 4.5, 4.6 or4.7) to about 5.0. In one aspect, the mixture is quenched by loweringthe pH of the mixture to 4.8. In another aspect, the mixture is quenchedby lowering the pH of the mixture to 5.5.

In one aspect, the one-step process further comprises a holding step torelease the unstably bound linkers from the antibody. The holding stepcomprises holding the mixture prior to purification of the conjugate(e.g., after the reaction step, between the reaction step and thequenching step, or after the quenching step). For example, the processcomprises (a) contacting the antibody with the drug (e.g., DM1 or DM4)to form a mixture comprising the antibody and the drug (e.g., DM1 orDM4); and then contacting the mixture comprising the antibody and drug(e.g., DM1 or DM4) with the cross-linking agent (e.g., SMCC, Sulfo-SMCC,SPDB, Sulfo-SPDB or CX1-1), in a solution having a pH of about 4 toabout 9 to provide a mixture comprising (i) the conjugate (e.g.,Ab-SMCC-DM1, Ab-SPDB-DM4 or Ab-CX1-1-DM1), (ii) free drug (e.g., DM1 orDM4), and (iii) reaction by-products, (b) holding the mixture preparedin step (a) to release the unstably bound linkers from the cell-bindingagent, and (c) purifying the mixture to provide a purified conjugate.

In another aspect, the process comprises (a) contacting the antibodywith the drug (e.g., DM1 or DM4) to form a mixture comprising theantibody and the drug (e.g., DM1 or DM4); and then contacting themixture comprising the antibody and the drug (e.g., DM1 or DM4) with thecross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1),in a solution having a pH of about 4 to about 9 to provide a mixturecomprising (i) the conjugate, (ii) free drug (e.g., DM1 or DM4), and(iii) reaction by-products, (b) quenching the mixture prepared in step(a) to quench any unreacted drug (e.g., DM1 or DM4) and/or unreactedcross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1),(c) holding the mixture prepared in step (b) to release the unstablybound linkers from the cell-binding agent, and (d) purifying the mixtureto provide a purified conjugate (e.g., Ab-SMCC-DM1, Ab-SPDB-DM4 orAb-CX1-1-DM1).

Alternatively, the holding step can be performed after purification ofthe conjugate, followed by an additional purification step.

In another aspect, the reaction is allowed to proceed to completionprior to the holding step. In this regard, the holding step can beperformed about 1 hour to about 48 hours (e.g., about 1 hour, about 2hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours,about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours,about 21 hours, about 22 hours, about 23 hours, about 24 hours, or about24 hours to about 48 hours) after the mixture comprising the antibodyand the drug (e.g., DM1 or DM4) is contacted with the cross-linkingagent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1).

The holding step comprises maintaining the solution at a suitabletemperature (e.g., about 0° C. to about 37° C.) for a suitable period oftime (e.g., about 1 hour to about 1 week, about 1 hour to about 24hours, about 1 hour to about 8 hours, or about 1 hour to about 4 hours)to release the unstably bound linkers from the antibody while notsubstantially releasing the stably bound linkers from the antibody. Inone aspect, the holding step comprises maintaining the solution at about20° C. or less (e.g., about 0° C. to about 18° C., about 4° C. to about16° C.), at room temperature (e.g., about 20° C. to about 30° C. orabout 20° C. to about 25° C.), or at an elevated temperature (e.g.,about 30° C. to about 37° C.). In one aspect, the holding step comprisesmaintaining the solution at a temperature of about 16° C. to about 24°C. (e.g., about 15° C., about 16° C., about 17° C., about 18° C., about19° C., about 20° C., about 21° C., about 22° C., about 23° C., about24° C., or about 25° C.). In another aspect, the holding step comprisesmaintaining the solution at a temperature of about 2° C. to about 8° C.(e.g., about 0° C., about 1° C., about 2° C., about 3° C., about 4° C.,about 5° C., about 6° C., about 7° C., about 8° C., about 9° C., orabout 10° C.). In another aspect, the holding step comprises maintainingthe solution at a temperature of about 37° C. (e.g., about 34° C., about35° C., about 36° C., about 37° C., about 38° C., about 39° C., or about40° C.).

The duration of the holding step depends on the temperature and the pHat which the holding step is performed. For example, the duration of theholding step can be substantially reduced by performing the holding stepat elevated temperature, with the maximum temperature limited by thestability of the cell-binding agent-cytotoxic agent conjugate. Theholding step can comprise maintaining the solution for about 1 hour toabout 1 day (e.g., about 1 hour, about 2 hours, about 3 hours, about 4hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about9 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours,about 18 hours, about 20 hours, about 22 hours, or about 24 hours),about 10 hours to about 24 hours, about 12 hours to about 24 hours,about 14 hours to about 24 hours, about 16 hours to about 24 hours,about 18 hours to about 24 hours, about 20 hours to about 24 hours,about 5 hours to about 1 week, about 20 hours to about 1 week, about 12hours to about 1 week (e.g., about 12 hours, about 16 hours, about 20hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5days, about 6 days, or about 7 days), or about 1 day to about 1 week.

In one aspect, the holding step comprises maintaining the solution at atemperature of about 2° C. to about 8° C. for a period of at least about12 hours for up to a week. In another aspect, the holding step comprisesmaintaining the solution at a temperature of about 2° C. to about 8° C.overnight (e.g., about 12 to about 24 hours, preferably about 20 hours).

The pH value for the holding step preferably is about 4 to about 10. Inone aspect, the pH value for the holding step is about 4 or more, butless than about 6 (e.g., 4 to 5.9) or about 5 or more, but less thanabout 6 (e.g., 5 to 5.9). In another aspect, the pH values for theholding step range from about 6 to about 10 (e.g., about 6.5 to about 9,about 6 to about 8). For example, pH values for the holding step can beabout 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9,about 9.5, or about 10.

In other aspects, the holding step can comprise incubating the mixtureat 25° C. at a pH of about 6-7.5 for about 12 hours to about 1 week,incubating the mixture at 4° C. at a pH of about 4.5-5.9 for about 5hours to about 5 days, or incubating the mixture at 25° C. at a pH ofabout 4.5-5.9 for about 5 hours to about 1 day.

The one-step process can optionally include the addition of sucrose tothe reaction step to increase solubility and recovery of the conjugates.Desirably, sucrose is added at a concentration of about 0.1% (w/v) toabout 20% (w/v) (e.g., about 0.1% (w/v), 1% (w/v), 5% (w/v), 10% (w/v),15% (w/v), or 20% (w/v)). Preferably, sucrose is added at aconcentration of about 1% (w/v) to about 10% (w/v) (e.g., about 0.5%(w/v), about 1% (w/v), about 1.5% (w/v), about 2% (w/v), about 3% (w/v),about 4% (w/v), about 5% (w/v), about 6% (w/v), about 7% (w/v), about 8%(w/v), about 9% (w/v), about 10% (w/v), or about 11% (w/v)). Inaddition, the reaction step also can comprise the addition of abuffering agent. Any suitable buffering agent known in the art can beused. Suitable buffering agents include, for example, a citrate buffer,an acetate buffer, a succinate buffer, and a phosphate buffer. In oneaspect, the buffering agent is selected from the group consisting ofHEPPSO(N-(2-hydroxyethyl)piperazine-N′-(2-hydroxypropanesulfonic acid)),POPSO (piperazine-1,4-bis-(2-hydroxy-propane-sulfonic acid) dehydrate),HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid), HEPPS (EPPS)(4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid), TES(N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid), and acombination thereof.

The one-step process can further comprise the step of purifying themixture to provide purified conjugate (e.g., Ab-SMCC-DM1, Ab-SPDB-DM4 orAb-CX1-1-DM1). Any purification methods known in the art can be used topurify the conjugates of the present disclosure. In one aspect, theconjugates of the present disclosure use tangential flow filtration(TFF), non-adsorptive chromatography, adsorptive chromatography,adsorptive filtration, selective precipitation, or any other suitablepurification process, as well as combinations thereof. In anotheraspect, prior to subjecting the conjugates to purification processdescribed above, the conjugates are first filtered through one or morePVDF membranes. Alternatively, the conjugates are filtered through oneor more PVDF membranes after subjecting the conjugates to thepurification process described above. For example, in one aspect, theconjugates are filtered through one or more PVDF membranes and thenpurified using tangential flow filtration. Alternatively, the conjugatesare purified using tangential flow filtration and then filtered throughone or more PVDF membranes.

Any suitable TFF systems may be utilized for purification, including aPellicon® type system (Millipore, Billerica, Mass.), a Sartocon®Cassette system (Sartorius AG, Edgewood, N.Y.), and a Centrasette® typesystem (Pall Corp., East Hills, N.Y.).

Any suitable adsorptive chromatography resin may be utilized forpurification. Preferred adsorptive chromatography resins includehydroxyapatite chromatography, hydrophobic charge inductionchromatography (HCIC), hydrophobic interaction chromatography (HIC), ionexchange chromatography, mixed mode ion exchange chromatography,immobilized metal affinity chromatography (IMAC), dye ligandchromatography, affinity chromatography, reversed phase chromatography,and combinations thereof. Examples of suitable hydroxyapatite resinsinclude ceramic hydroxyapatite (CHT Type I and Type II, Bio-RadLaboratories, Hercules, Calif.), HA Ultrogel® hydroxyapatite (PallCorp., East Hills, N.Y.), and ceramic fluoroapatite (CFT Type I and TypeII, Bio-Rad Laboratories, Hercules, Calif.). An example of a suitableHCIC resin is MEP Hypercel® resin (Pall Corp., East Hills, N.Y.).Examples of suitable HIC resins include Butyl-Sepharose,Hexyl-Sepaharose, Phenyl-Sepharose, and Octyl Sepharose resins (all fromGE Healthcare, Piscataway, N.J.), as well as Macro-prep® Methyl andMacro-Prep® t-Butyl resins (Biorad Laboratories, Hercules, Calif.).Examples of suitable ion exchange resins include SP-Sepharose®,Sepharoset, and Q-Sepharose® resins (all from GE Healthcare, Piscataway,N.J.), and Unosphere® S resin (Bio-Rad Laboratories, Hercules, Calif.).Examples of suitable mixed mode ion exchangers include Bakerbond® ABxresin (JT Baker, Phillipsburg N.J.). Examples of suitable IMAC resinsinclude Chelating Sepharose® resin (GE Healthcare, Piscataway, N.J.) andProfinity® IMAC resin (Bio-Rad Laboratories, Hercules, Calif.). Examplesof suitable dye ligand resins include Blue Sepharose resin (GEHealthcare, Piscataway, N.J.) and Affi-gel Blue resin (Bio-RadLaboratories, Hercules, Calif.). Examples of suitable affinity resinsinclude Protein A Sepharose resin (e.g., MabSelect, GE Healthcare,Piscataway, N.J.) and lectin affinity resins, e.g. Lentil LectinSepharose® resin (GE Healthcare, Piscataway, N.J.), where the antibodybears appropriate lectin binding sites. Examples of suitable reversedphase resins include C4, C8, and C18 resins (Grace Vydac, Hesperia,Calif.).

Any suitable non-adsorptive chromatography resin may be utilized forpurification. Examples of suitable non-adsorptive chromatography resinsinclude, but are not limited to, SEPHADEX™ G-25, G-50, G-100, SEPHACRYL™resins (e.g., S-200 and S-300), SUPERDEX™ resins (e.g., SUPERDEX™ 75 andSUPERDEX™ 200), BIO-GEL® resins (e.g., P-6, P-10, P-30, P-60, andP-100), and others known to those of ordinary skill in the art.

Two-Step Process and One-Pot Process

In one aspect, the conjugates of the present disclosure can be preparedas described in the U.S. Pat. No. 7,811,572 and U.S. Patent ApplicationPublication No. 2006/0182750. The process comprises the steps of (a)contacting the antibody of the present disclosure with the cross-linkingagent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1) to covalentlyattach the linker (i.e., Ab-SMCC, Ab-SPDB or Ab-CX1-1) to the antibodyand thereby prepare a first mixture comprising the antibody having thelinker bound thereto; (b) optionally subjecting the first mixture to apurification process to prepare a purified first mixture of the antibodyhaving the linker bound thereto; (c) conjugating the drug (e.g., DM1 orDM4) to the antibody having the linker bound thereto in the firstmixture by reacting the antibody having the linker bound thereto withthe drug (e.g., DM1 or DM4) in a solution having a pH of about 4 toabout 9 to prepare a second mixture comprising (i) conjugate (e.g.,Ab-SMCC-DM1, Ab-SPDB-DM4 or Ab-CX1-1-DM1), (ii) free drug (e.g., DM1 orDM4); and (iii) reaction by-products; and (d) subjecting the secondmixture to a purification process to purify the conjugate from the othercomponents of the second mixture. Alternatively, the purification step(b) can be omitted. Any purification methods described herein can beused for steps (b) and (d). In one embodiment, TFF is used for bothsteps (b) and (d). In another embodiment, TFF is used for step (b) andabsorptive chromatography (e.g., CHT) is used for step (d).

One-Step Reagent and In-situ Process

In one aspect, the conjugates of the present disclosure can be preparedby conjugating pre-formed drug-linker compound (e.g., SMCC-DM1,Sulfo-SMCC-DM1, SPDB-DM4 or CX1-1-DM1) to the antibody of the presentdisclosure, as described in U.S. Pat. No. 6,441,163 and U.S. PatentApplication Publication Nos. 2011/0003969 and 2008/0145374, followed bya purification step. Any purification methods described herein can beused. The drug-linker compound is prepared by reacting the drug (e.g.,DM1 or DM4) with the cross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB,Sulfo-SPDB or CX1-1). The drug-linker compound (e.g., SMCC-DM1,Sulfo-SMCC-DM1, SPDB-DM4 or CX1-1-DM1) is optionally subjected topurification before being conjugated to the antibody.

4. Characterization and Selection of Desirable Antibodies and AntibodyDrug-Conjugates

The antibodies, antibody fragments (e.g., antigen binding fragments) orantibody drug conjugates of the present disclosure can be characterizedand selected for their physical/chemical properties and/or biologicalactivities by various assays known in the art.

For example, an antibody of the present disclosure can be tested for itsantigen binding activity by known methods such as ELISA, FACS, Biacoreor Western blot.

Transgenic animals and cell lines are particularly useful in screeningantibody drug conjugates (ADCs) that have potential as prophylactic ortherapeutic treatments of cancer overexpression of tumor-associatedantigens and cell surface receptors. Screening for a useful ADC mayinvolve administering a candidate ADC over a range of doses to thetransgenic animal, and assaying at various time points for the effect(s)of the ADC on the disease or disorder being evaluated. Alternatively, oradditionally, the drug can be administered prior to or simultaneouslywith exposure to an inducer of the disease, if applicable. The candidateADC may be screened serially and individually, or in parallel undermedium or high-throughput screening format.

One aspect is a screening method comprising (a) transplanting cells froma stable cancer cell line or human patient tumor expressing cKIT (e.g.,a GIST cell line or tumor fragment, a melanoma cell line or tumorfragment) into a non-human animal, (b) administering an ADC drugcandidate to the non-human animal and (c) determining the ability of thecandidate to inhibit the growth of tumors from the transplanted cellline. The present disclosure also encompasses a method of screening ADCcandidates for the treatment of a disease or disorder characterized bythe overexpression of cKIT comprising (a) contacting cells from a stablecancer cell line expressing cKIT with a drug candidate, and (b)evaluating the ability of the ADC candidate to inhibit the growth of thestable cell line.

Another aspect is a screening method comprising (a) contacting cellsfrom a stable cancer cell line expressing cKIT with an ADC drugcandidate and (b) evaluating the ability of the ADC candidate to blockligand activation of cKIT. In another aspect the ability of the ADCcandidate to block ligand-stimulated tyrosine phosphorylation isevaluated.

A further aspect is a screening method comprising (a) contacting cellsfrom a stable cancer cell line expressing cKIT with an ADC drugcandidate and (b) evaluating the ability of the ADC candidate to inducecell death. In one aspect the ability of the ADC candidate to induceapoptosis is evaluated.

Candidate ADC can be screened by being administered to the transgenicanimal over a range of doses, and evaluating the animal's physiologicalresponse to the compounds over time. In some cases, it can beappropriate to administer the compound in conjunction with co-factorsthat would enhance the efficacy of the compound. If cell lines derivedfrom the subject transgenic animals are used to screen for ADCs usefulin treating various disorders associated with overexpression of cKIT,the test ADCs are added to the cell culture medium at an appropriatetime, and the cellular response to the ADCs is evaluated over time usingthe appropriate biochemical and/or histological assays.

Thus, the present disclosure provides assays for identifying ADC whichspecifically target and bind to cKIT, and cKIT overexpression on tumorcells.

cKIT Antibodies

The present disclosure provides for antibodies or antibody fragments(e.g., antigen binding fragments) that specifically bind to human cKIT.Antibodies or antibody fragments (e.g., antigen binding fragments) ofthe present disclosure include, but are not limited to, the humanmonoclonal antibodies or fragments thereof, isolated as described, inthe Examples below.

The present disclosure in certain aspects provides antibodies orantibody fragments (e.g., antigen binding fragments) that specificallybind cKIT, said antibodies or antibody fragments (e.g., antigen bindingfragments) comprise a VH domain having an amino acid sequence of SEQ IDNO: 9, 28, 46, 64, 82, 100, 118 or 136 (Table 1). The present disclosurealso provides antibodies or antibody fragments (e.g., antigen bindingfragments) that specifically bind to cKIT, said antibodies or antibodyfragments (e.g., antigen binding fragments) comprise a VH CDR having anamino acid sequence of any one of the VH CDRs listed in Table 1. Inparticular aspects, the present disclosure provides antibodies orantibody fragments (e.g., antigen binding fragments) that specificallybind to cKIT, said antibodies comprising (or alternatively, consist of)one, two, three, four, five or more VH CDRs having an amino acidsequence of any of the VH CDRs listed in Table 1, infra.

The present disclosure provides antibodies or antibody fragments (e.g.,antigen binding fragments) that specifically bind to cKIT, saidantibodies or antibody fragments (e.g., antigen binding fragments)comprise a VL domain having an amino acid sequence of SEQ ID NO: 18, 37,55, 73, 91, 109, 127 or 145 (Table1). The present disclosure alsoprovides antibodies or antibody fragments (e.g., antigen bindingfragments) that specifically bind to cKIT, said antibodies or antibodyfragments (e.g., antigen binding fragments) comprise a VL CDR having anamino acid sequence of any one of the VL CDRs listed in Table 1, infra.In particular, the disclosure provides antibodies or antibody fragments(e.g., antigen binding fragments) that specifically bind to cKIT, saidantibodies or antibody fragments (e.g., antigen binding fragments)comprise (or alternatively, consist of) one, two, three or more VL CDRshaving an amino acid sequence of any of the VL CDRs listed in Table 1.

Other antibodies or antibody fragments (e.g., antigen binding fragments)of the present disclosure include amino acids that have been mutated,yet have at least 60, 70, 80, 90 or 95 percent identity in the CDRregions with the CDR regions depicted in the sequences described inTable 1. In some aspects, it includes mutant amino acid sequenceswherein no more than 1, 2, 3, 4 or 5 amino acids have been mutated inthe CDR regions when compared with the CDR regions depicted in thesequence described in Table 1.

The present disclosure also provides nucleic acid sequences that encodeVH, VL, the full length heavy chain, and the full length light chain ofthe antibodies that specifically bind to cKIT. Such nucleic acidsequences can be optimized for expression in mammalian cells.

TABLE 1 Examples of anti- cKIT Antibodies 9P3 SEQ ID NO 3: (Kabat) HCDR1DYYMA SEQ ID NO 4: (Kabat) HCDR2 NINYDGSSTYYLDSLKS SEQ ID NO 5: (Kabat)HCDR3 GDYYGTTYWYFDV SEQ ID NO 6: (Chothia) HCDR1 GFTFSDYSEQ ID NO 7: (Chothia) HCDR2 NYDGSS SEQ ID NO 8: (Chothia) HCDR3GDYYGTTYWYFDV SEQ ID NO 9: VHEVRLVESEGGLVQPRSSMKLSCTASGFTFSDYYMAWVRQVPEKGLEWVANINYDGSSTYYLDSLKSRFIISRDNAKNILYLQMSSLKSEDTATYYCARGDYYGTTYWYFDVWGTGTTVTVSS SEQ ID NO 10 ConstantVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL heavyQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ chain GLSSPVTKSFNRGECSEQ ID NO 11: Heavy EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMAWVRQAPGKG ChainLEWVANINYDGSSTYYLDSVKGRFTISRDNAKNSLYLQMNSLRA (humanized)EDTAVYYCARGDYYGTTYWYFDVWGQGTTYTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKSEQ ID NO 12: (Kabat) LCDR1 RASQDISNYLN SEQ ID NO 13: (Kabat) LCDR2YTSRLQS SEQ ID NO 14: (Kabat) LCDR3 QQGKKLWS SEQ ID NO 15: LCDR1 SQDISNY(Chothia) SEQ ID NO 16: LCDR2 YTS (Chothia) SEQ ID NO 17: LCDR3 GKKLW(Chothia) SEQ ID NO 18: VL DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLQSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGK KLWSFGGGTKLEIKRSEQ ID NO:19 Constant ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAlight LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN chainTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVELFTPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGKSEQ ID NO 20: Light DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKL ChainLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGK (humanizedKLWSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF VK1)YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO 21: DNAEIVMTQSPATLSLSPGERATLSCRASQDISNYLNWYQQKPGQAPR LightLLIYYTSRLQSGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQGK ChainKLWSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF (humanizedYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK VK3ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC NEG009) NEG024 SEQ ID NO 22: (Kabat)HCDR1 DYYMA SEQ ID NO 23: (Kabat) HCDR2 NINQIAGSTYYLDSVRGSEQ ID NO 24: (Kabat) HCDR3 GDYYGTTYWYFDV SEQ ID NO 25: HCDR1 GFTFSDY(Chothia) SEQ ID NO 26: HCDR2 NQIAGS (Chothia) SEQ ID NO 27: HCDR3GDYYGTTYWYFDV (Chothia) SEQ ID NO 28: VHEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMAWVRQAPGKGLEWVANINQIAGSTYYLDSVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGDYYGTTYWYFDVWGQGTTVTVSS SEQ ID NO 29: HeavyEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMAWVRQAPGKG ChainLEWVANINQIAGSTYYLDSVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGDYYGTTYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK SEQ ID NO 30: DNAGAAGTGCAATTGGTGGAAAGCGGCGGAGGCCTGGTGCAGCCT HeavyGGCGGCTCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCT ChainTCAGCGACTACTACATGGCCTGGGTCCGACAGGCCCCTGGCAAGGGCCTGGAATGGGTGGCCAATATCAACCAAATCGCCGGCAGCACCTACTACCTGGACAGCGTGAGAGGCCGGTTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGAGGCGATTACTACGGCACCACCTACTGGTACTTCGACGTGTGGGGCCAGGGCACCACCGTGACCGTCAGCTCAGCTAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGTCCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCCCCAGAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAAGGTCTCCAACAAGGCCCTGCCAGCCCCCATCGAAAAGACCATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCTCCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCCCGGCAAG SEQ ID NO 31: (Kabat) LCDR1 RASQDISNYLNSEQ ID NO 32: (Kabat) LCDR2 YTSRLQS SEQ ID NO 33: (Kabat) LCDR3 QQGKKLWSSEQ ID NO 34: LCDR1 SQDISNY (Chothia) SEQ ID NO 35: LCDR2 YTS (Chothia)SEQ ID NO 36: LCDR3 GKKLW (Chothia) SEQ ID NO 37: VLEIVMTQSPATLSLSPGERATLSCRASQDISNYLNWYQQKPGQAPRLLIYYTSRLQSGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQGK KLWSFGGGTKVEIKSEQ ID NO 38: Light EIVMTQSPATLSLSPGERATLSCRASQDISNYLNWYQQKPGQAPR ChainLLIYYTSRLQSGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQGKKLWSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO 39: DNAGAGATCGTGATGACCCAGAGCCCCGCCACCCTGAGCCTGAGCC LightCTGGCGAAAGAGCCACCCTGTCCTGCAGAGCCAGCCAGGACAT ChainCAGCAACTACCTGAACTGGTATCAGCAGAAGCCCGGCCAGGCCCCCAGACTGCTGATCTACTACACCAGCCGGCTGCAGAGCGGCATCCCCGCCAGATTTTCTGGCAGCGGCAGCGGCACCGACTACACCCTGACCATCAGCAGCCTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGGCAAGAAGCTGTGGTCCTTCGGCGGAGGCACCAAGGTGGAAATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC NEG026 SEQ ID NO 40: (Kabat)HCDR1 DYYMA SEQ ID NO 41: (Kabat) HCDR2 NINQNTGSTYYVDSVQGSEQ ID NO 42: (Kabat) HCDR3 GDYYGTTYWYFDV SEQ ID NO 43: HCDR1 GFTFSDY(Chothia) SEQ ID NO 44: HCDR2 NQNTGS (Chothia) SEQ ID NO 45: HCDR3GDYYGTTYWYFDV (Chothia) SEQ ID NO 46: VHEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMAWVRQAPGKGLEWVANINQNTGSTYYVDSVQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGDYYGTTYWYFDVWGQGTTVTVSS SEQ ID NO 47: HeavyEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMAWVRQAPGKG ChainLEWVANINQNTGSTYYVDSVQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGDYYGTTYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQ ID NO 48: DNAGAAGTGCAATTGGTGGAAAGCGGCGGAGGCCTGGTGCAGCCT HeavyGGCGGCTCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCT ChainTCAGCGACTACTACATGGCCTGGGTCCGACAGGCCCCTGGCAAGGGCCTGGAATGGGTGGCCAATATCAACCAAAACACCGGCAGCACCTACTACGTGGACAGCGTGCAAGGCCGGTTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGAGGCGATTACTACGGCACCACCTACTGGTACTTCGACGTGTGGGGCCAGGGCACCACCGTGACCGTCAGCTCAGCTAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGTCCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCCCCAGAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAAGGTCTCCAACAAGGCCCTGCCAGCCCCCATCGAAAAGACCATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCTCCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCCCGGCAAG SEQ ID NO 49: (Kabat) LCDR1 RASQDISNYLNSEQ ID NO 50: (Kabat) LCDR2 YTSRLQS SEQ ID NO 51: (Kabat) LCDR3 QQGKKLWSSEQ ID NO 52: LCDR1 SQDISNY (Chothia) SEQ ID NO 53: LCDR2 YTS (Chothia)SEQ ID NO 54: LCDR3 GKKLW (Chothia) SEQ ID NO 55: VLEIVMTQSPATLSLSPGERATLSCRASQDISNYLNWYQQKPGQAPRLLIYYTSRLQSGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQGK KLWSFGGGTKVEIKSEQ ID NO 56: Light EIVMTQSPATLSLSPGERATLSCRASQDISNYLNWYQQKPGQAPR ChainLLIYYTSRLQSGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQGKKLWSFGGGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC SEQ ID NO 57: DNAGAGATCGTGATGACCCAGAGCCCCGCCACCCTGAGCCTGAGCC LightCTGGCGAAAGAGCCACCCTGTCCTGCAGAGCCAGCCAGGACAT ChainCAGCAACTACCTGAACTGGTATCAGCAGAAGCCCGGCCAGGCCCCCAGACTGCTGATCTACTACACCAGCCGGCTGCAGAGCGGCATCCCCGCCAGATTTTCTGGCAGCGGCAGCGGCACCGACTACACCCTGACCATCAGCAGCCTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGGCAAGAAGCTGTGGTCCTTCGGCGGAGGCACCAAGGTGGAAATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC NEG027 SEQ ID NO 58: (Kabat)HCDR1 DYYMA SEQ ID NO 59: (Kabat) HCDR2 SINQNTGSTYYLDSVRGSEQ ID NO 60: (Kabat) HCDR3 GDYYGTTYWYFDV SEQ ID NO 61: HCDR1 GFTFSDY(Chothia) SEQ ID NO 62: HCDR2 NQNTGS (Chothia) SEQ ID NO 63: HCDR3GDYYGTTYWYFDV (Chothia) SEQ ID NO 64: VHEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMAWVRQAPGKGLEWVASINQNTGSTYYLDSVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGDYYGTTYWYFDVWGQGTTVTVSS SEQ ID NO 65: HeavyEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMAWVRQAPGKG ChainLEWVASINQNTGSTYYLDSVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGDYYGTTYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK SEQ ID NO 66: DNAGAAGTGCAATTGGTGGAAAGCGGCGGAGGCCTGGTGCAGCCT HeavyGGCGGCTCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCT ChainTCAGCGACTACTACATGGCCTGGGTCCGACAGGCCCCTGGCAAGGGCCTGGAATGGGTGGCCAGTATCAACCAAAACACCGGCAGCACCTACTACCTGGACAGCGTGCGAGGCCGGTTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGAGGCGATTACTACGGCACCACCTACTGGTACTTCGACGTGTGGGGCCAGGGCACCACCGTGACCGTCAGCTCAGCTAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGTCCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCCCCAGAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAAGGTCTCCAACAAGGCCCTGCCAGCCCCCATCGAAAAGACCATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCTCCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCCCGGCAAG SEQ ID NO 67: (Kabat) LCDR1 RASQDISNYLNSEQ ID NO 68: (Kabat) LCDR2 YTSRLQS SEQ ID NO 69: (Kabat) LCDR3 QQGKKLWSSEQ ID NO 70: LCDR1 SQDISNY (Chothia) SEQ ID NO 71: LCDR2 YTS (Chothia)SEQ ID NO 72: LCDR3 GKKLW (Chothia) SEQ ID NO 73: VLEIVMTQSPATLSLSPGERATLSCRASQDISNYLNWYQQKPGQAPRLLIYYTSRLQSGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQGK KLWSFGGGTKVEIKSEQ ID NO 74: Light EIVMTQSPATLSLSPGERATLSCRASQDISNYLNWYQQKPGQAPR ChainLLIYYTSRLQSGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQGKKLWSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO 75: DNAGAGATCGTGATGACCCAGAGCCCCGCCACCCTGAGCCTGAGCC LightCTGGCGAAAGAGCCACCCTGTCCTGCAGAGCCAGCCAGGACAT ChainCAGCAACTACCTGAACTGGTATCAGCAGAAGCCCGGCCAGGCCCCCAGACTGCTGATCTACTACACCAGCCGGCTGCAGAGCGGCATCCCCGCCAGATTTTCTGGCAGCGGCAGCGGCACCGACTACACCCTGACCATCAGCAGCCTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGGCAAGAAGCTGTGGTCCTTCGGCGGAGGCACCAAGGTGGAAATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC NEG085 SEQ ID NO 76: (Kabat)HCDR1 GYYMA SEQ ID NO 77: (Kabat) HCDR2 NINYPGSSTYYLDSVKGSEQ ID NO 78: (Kabat) HCDR3 GDYYGTTYWYFDV SEQ ID NO 79: HCDR1 GFAFSGY(Chothia) SEQ ID NO 80: HCDR2 NYPGSS (Chothia) SEQ ID NO 81: HCDR3GDYYGTTYWYFDV (Chothia) SEQ ID NO 82: VHEVQLVESGGGLVQPGGSLRLSCAASGFAFSGYYMAWVRQAPGKGLEWVANINYPGSSTYYLDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGDYYGTTYWYFDVWGQGTTVTVSS SEQ ID NO 83: HeavyEVQLVESGGGLVQPGGSLRLSCAASGFAFSGYYMAWVRQAPGK ChainGLEWVANINYPGSSTYYLDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGDYYGTTYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQ ID NO 84: DNAGAAGTGCAATTGGTGGAAAGCGGCGGAGGCCTGGTGCAGCCT HeavyGGCGGCTCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCGCCT ChainTCAGCGGCTACTACATGGCCTGGGTCCGACAGGCCCCTGGCAAGGGCCTGGAATGGGTGGCCAACATCAACTACCCCGGCAGCAGCACCTACTACCTGGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGAGGCGATTACTACGGCACCACCTACTGGTACTTCGACGTGTGGGGCCAGGGCACCACCGTGACCGTCAGCTCAGCTAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGTCCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCCCCAGAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAAGGTCTCCAACAAGGCCCTGCCAGCCCCCATCGAAAAGACCATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCTCCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCCCGGCAAG SEQ ID NO 85: (Kabat) LCDR1 RASQSISSYLNSEQ ID NO 86: (Kabat) LCDR2 YTSRLQS SEQ ID NO 87: (Kabat) LCDR3 QQGRRLWSSEQ ID NO 88: LCDR1 SQSISSY (Chothia) SEQ ID NO 89: LCDR2 YTS (Chothia)SEQ ID NO 90: LCDR3 GRRLW (Chothia) SEQ ID NO 91: VLDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGRR LWSFGGGTKVEIKSEQ ID NO 92: Light DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKL ChainLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGRRLWSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO 93: DNAGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCG LightTGGGCGACAGAGTGACCATCACCTGTCGGGCCAGCCAGAGCAT ChainCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACTACACCAGCCGGCTGCAGAGCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGGCCGCCGCCTGTGGTCCTTCGGCGGAGGCACCAAGGTGGAAATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC NEG086 SEQ ID NO 94: (Kabat)HCDR1 DYYMA SEQ ID NO 95: (Kabat) HCDR2 NINQIAGSTYYVDSVQGSEQ ID NO 96: (Kabat) HCDR3 GDYYGTTYWYFDV SEQ ID NO 97: HCDR1 GFTFSDY(Chothia) SEQ ID NO 98: HCDR2 NQIAGS (Chothia) SEQ ID NO 99: HCDR3GDYYGTTYWYFDV (Chothia) SEQ ID NO 100: VHEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMAWVRQAPGKGLEWVANINQIAGSTYYVDSVQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGDYYGTTYWYFDVWGQGTTVTVSS SEQ ID NO 101: HeavyEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMAWVRQAPGKG ChainLEWVANINQIAGSTYYVDSVQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGDYYGTTYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK SEQ ID NO 102: DNAGAAGTGCAATTGGTGGAAAGCGGCGGAGGCCTGGTGCAGCCT HeavyGGCGGCTCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCT ChainTCAGCGACTACTACATGGCCTGGGTCCGACAGGCCCCTGGCAAGGGCCTGGAATGGGTGGCCAATATCAACCAAATCGCCGGCAGCACCTACTACGTGGACAGCGTGCAAGGCCGGTTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGAGGCGATTACTACGGCACCACCTACTGGTACTTCGACGTGTGGGGCCAGGGCACCACCGTGACCGTCAGCTCAGCTAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGTCCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCCCCAGAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAAGGTCTCCAACAAGGCCCTGCCAGCCCCCATCGAAAAGACCATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCTCCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCCCGGCAAG SEQ ID NO 103: (Kabat) LCDR1 RASQSISSYLNSEQ ID NO 104: (Kabat) LCDR2 YTSRLQS SEQ ID NO 105: (Kabat) LCDR3QQGRRLWS SEQ ID NO 106: LCDR1 SQSISSY (Chothia) SEQ ID NO 107: LCDR2 YTS(Chothia) SEQ ID NO 108: LCDR3 GRRLW (Chothia) SEQ ID NO 109: VLDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGRR LWSFGGGTKVEIKSEQ ID NO 110: Light DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLChain LIYYTSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGRRLWSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO 111: DNAGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCG LightTGGGCGACAGAGTGACCATCACCTGTCGGGCCAGCCAGAGCAT ChainCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACTACACCAGCCGGCTGCAGAGCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGGCCGCCGCCTGTGGTCCTTCGGCGGAGGCACCAAGGTGGAAATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC NEG087 SEQ ID NO 112: (Kabat)HCDR1 DYYMA SEQ ID NO 113: (Kabat) HCDR2 SINQNTGSTYYLDSVRGSEQ ID NO 114: (Kabat) HCDR3 GDYYGTTYWYFDV SEQ ID NO 115: HCDR1 GFTFSDY(Chothia) SEQ ID NO 116: HCDR2 NQNTGS (Chothia) SEQ ID NO 117: HCDR3GDYYGTTYWYFDV (Chothia) SEQ ID NO 118: VHEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMAWVRQAPGKGLEWVASINQNTGSTYYLDSVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGDYYGTTYWYFDVWGQGTTVTVSS SEQ ID NO 119: HeavyEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMAWVRQAPGKG ChainLEWVASINQNTGSTYYLDSVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGDYYGTTYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK SEQ ID NO 120: DNAGAAGTGCAATTGGTGGAAAGCGGCGGAGGCCTGGTGCAGCCT HeavyGGCGGCTCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCT ChainTCAGCGACTACTACATGGCCTGGGTCCGACAGGCCCCTGGCAAGGGCCTGGAATGGGTGGCCAGTATCAACCAAAACACCGGCAGCACCTACTACCTGGACAGCGTGCGAGGCCGGTTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGAGGCGATTACTACGGCACCACCTACTGGTACTTCGACGTGTGGGGCCAGGGCACCACCGTGACCGTCAGCTCAGCTAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGTCCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCCCCAGAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAAGGTCTCCAACAAGGCCCTGCCAGCCCCCATCGAAAAGACCATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCTCCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCCCGGCAAG SEQ ID NO 121: (Kabat) LCDR1RASQSISSYLN SEQ ID NO 122: (Kabat) LCDR2 YTSRLQS SEQ ID NO 123: (Kabat)LCDR3 QQGRRLWS SEQ ID NO 124: LCDR1 SQSISSY (Chothia) SEQ ID NO 125:LCDR2 YTS (Chothia) SEQ ID NO 126: LCDR3 GRRLW (Chothia) SEQ ID NO 127:VL DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGRR LWSFGGGTKVEIKSEQ ID NO 128: Light DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLChain LIYYTSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGRRLWSFGGGTKVEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO 129: DNAGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCG LightTGGGCGACAGAGTGACCATCACCTGTCGGGCCAGCCAGAGCAT ChainCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACTACACCAGCCGGCTGCAGAGCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGGCCGCCGCCTGTGGTCCTTCGGCGGAGGCACCAAGGTGGAAATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC 20376 SEQ ID NO 130: (Kabat)HCDR1 SYAIS SEQ ID NO 131: (Kabat) HCDR2 GIIPMSGRTTYAQKFQGSEQ ID NO 132: (Kabat) HCDR3 DYGPEAPDYGQSTSYFWYYAFDP SEQ ID NO 133:HCDR1 GGTFSSY (Chothia) SEQ ID NO 134: HCDR2 IPMSGR (Chothia)SEQ ID NO 135: HCDR3 DYGPEAPDYGQSTSYFWYYAFDP (Chothia) SEQ ID NO 136: VHQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPMSGRTTYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDYGPEAPDYGQSTSYFWYYAFDPWGQGTLVTVSS SEQ ID NO 137: HeavyQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQG ChainLEWMGGIIPMSGRTTYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDYGPEAPDYGQSTSYFWYYAFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGKSEQ ID NO 138: DNA CAGGTGCAGCTGGTGCAGTCAGGCGCCGAAGTGAAGAAACCC HeavyGGCTCTAGCGTGAAAGTCAGCTGTAAAGCTAGTGGGGGCACCT ChainTCTCTAGCTACGCTATTAGCTGGGTCAGACAGGCCCCAGGTCAAGGCTTGGAGTGGATGGGCGGAATTATCCCTATGAGCGGTAGAACTACCTACGCTCAGAAATTTCAGGGTAGAGTGACTATCACCGCCGACGAGTCTACTAGCACCGCCTATATGGAACTGAGTTCTCTGAGGTCAGAGGACACCGCCGTCTACTACTGCGCTAGAGACTACGGCCCCGAGGCCCCCGACTACGGTCAATCAACTAGCTACTTCTGGTACTACGCCTTCGACCCTTGGGGTCAAGGCACCCTGGTCACCGTGTCTTCAGCTAGCACTAAGGGCCCAAGTGTGTTTCCCCTGGCCCCCAGCAGCAAGTCTACTTCCGGCGGAACTGCTGCCCTGGGTTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCCTGGAACTCTGGGGCTCTGACTTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACAGTGCCCTCCAGCTCTCTGGGAACCCAGACCTATATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCTCCAGAACTGCTGGGAGGGCCTTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACAAGTGCAAAGTCTCCAACAAGGCCCTGCCAGCCCCAATCGAAAAGACAATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCAGCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCC TGAGCCCCGGCAAGSEQ ID NO 139: (Kabat) LCDR1 SGDNIPSYFVH SEQ ID NO 140: (Kabat) LCDR2DDNDRPS SEQ ID NO 141: (Kabat) LCDR3 SSWDQDTVV SEQ ID NO 142: LCDR1DNIPSYF (Chothia) SEQ ID NO 143: LCDR2 DDN (Chothia) SEQ ID NO 144:LCDR3 WDQDTV (Chothia) SEQ ID NO 145: VLDIELTQPPSVSVSPGQTASITCSGDNIPSYFVHWYQQKPGQAPVLVIYDDNDRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCSSWDQ DTVVFGGGTKLTVLSEQ ID NO 146: Light DIELTQPPSVSVSPGQTASITCSGDNIPSYFVHWYQQKPGQAPVLVIChain YDDNDRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCSSWDQDTVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS SEQ ID NO 147: DNAGATATCGAGCTGACTCAGCCCCCTAGCGTCAGCGTCAGCCCTG LightGTCAAACCGCCTCTATCACCTGTAGCGGCGATAATATCCCTAG ChainCTACTTCGTGCACTGGTATCAGCAGAAGCCCGGTCAAGCCCCCGTGCTGGTGATCTACGACGATAACGATAGACCTAGCGGAATCCCCGAGCGGTTTAGCGGCTCTAATAGCGGTAACACCGCTACCCTGACTATTAGCGGCACTCAGGCCGAGGACGAGGCCGACTACTACTGCTCTAGCTGGGATCAGGACACCGTGGTGTTCGGCGGAGGCACTAAGCTGACCGTGCTGGGTCAACCTAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAGCGAGGAGCTGCAGGCCAACAAGGCCACCCTGGTGTGCCTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCACCACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTGACCCACGAGGGCAGCACCGTGGAAAAGACCGTGGCCCCAACCGAGTGCAGC

Other antibodies of the present disclosure include those where the aminoacids or nucleic acids encoding the amino acids have been mutated, yethave at least 60, 70, 80, 90 or 95 percent identity to the sequencesdescribed in Table 1. In some aspects, it includes mutant amino acidsequences wherein no more than 1, 2, 3, 4 or 5 amino acids have beenmutated in the variable regions when compared with the variable regionsdepicted in the sequence described in Table 1, while retainingsubstantially the same therapeutic activity.

Since each of these antibodies can bind to cKIT, the VH, VL, full lengthlight chain, and full length heavy chain sequences (amino acid sequencesand the nucleotide sequences encoding the amino acid sequences) can be“mixed and matched” to create other cKIT-binding antibodies. Such “mixedand matched” cKIT-binding antibodies can be tested using the bindingassays known in the art (e.g., ELISAs, and other assays described in theExample section). When these chains are mixed and matched, a VH sequencefrom a particular VH/VL pairing should be replaced with a structurallysimilar VH sequence. Likewise a full length heavy chain sequence from aparticular full length heavy chain/full length light chain pairingshould be replaced with a structurally similar full length heavy chainsequence. Likewise, a VL sequence from a particular VH/VL pairing shouldbe replaced with a structurally similar VL sequence. Likewise, a fulllength light chain sequence from a particular full length heavychain/full length light chain pairing should be replaced with astructurally similar full length light chain sequence. Accordingly, inone aspect, the disclosure provides for an isolated monoclonal antibodyor antigen binding region thereof having: a heavy chain variable regioncomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 9, 28, 46, 64, 82, 100, 118 or 136 (Table 1); and a lightchain variable region comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 18, 37, 55, 73, 91, 109, 127 or 145(Table1); wherein the antibody specifically binds to cKIT.

In another aspect, the disclosure provides (i) an isolated monoclonalantibody having: a full length heavy chain comprising an amino acidsequence that has been optimized for expression in the cell of amammalian selected from the group consisting of SEQ ID NOs: 11, 29, 47,65, 83, 101, 119, or 137; and a full length light chain comprising anamino acid sequence that has been optimized for expression in the cellof a mammalian selected from the group consisting of SEQ ID NOs: 20, 21,38, 56, 74, 92, 110, 128, or 146; or (ii) a functional proteincomprising an antigen binding portion thereof.

In another aspect, the present disclosure provides cKIT-bindingantibodies that comprise the heavy chain and light chain CDR1s, CDR2sand CDR3s as described in Table 1, or combinations thereof. The aminoacid sequences of the VH CDR1s of the antibodies are shown in SEQ IDNOs: 3, 22, 40, 58, 76, 94, 112 and 130. The amino acid sequences of theVH CDR2s of the antibodies and are shown in SEQ ID NOs: 4, 23, 41, 59,77, 95, 113 and 131. The amino acid sequences of the VH CDR3s of theantibodies are shown in SEQ ID NOs: 5, 24, 42, 60, 78, 96, 114 and 132.The amino acid sequences of the VL CDR1s of the antibodies are shown inSEQ ID NOs: 12, 31, 49, 67, 85, 103, 121 and 139. The amino acidsequences of the VL CDR2s of the antibodies are shown in SEQ ID NOs 13,32, 50, 68, 86, 104, 122 and 140. The amino acid sequences of the VLCDR3s of the antibodies are shown in SEQ ID NOs:14, 33, 51, 69, 87, 105,123 and 141.

Given that each of these antibodies can bind to cKIT and thatantigen-binding specificity is provided primarily by the CDR1, 2 and 3regions, the VH CDR1, 2 and 3 sequences and VL CDR1, 2 and 3 sequencescan be “mixed and matched” (i.e., CDRs from different antibodies can bemixed and match, although each antibody must contain a VH CDR1, 2 and 3and a VL CDR1, 2 and 3 to create other CS-binding binding molecules.Such “mixed and matched” cKIT-binding antibodies can be tested using thebinding assays known in the art and those described in the Examples(e.g., ELISAs). When VH CDR sequences are mixed and matched, the CDR1,CDR2 and/or CDR3 sequence from a particular VH sequence should bereplaced with a structurally similar CDR sequence(s). Likewise, when VLCDR sequences are mixed and matched, the CDR1, CDR2 and/or CDR3 sequencefrom a particular VL sequence should be replaced with a structurallysimilar CDR sequence(s). It will be readily apparent to the ordinarilyskilled artisan that novel VH and VL sequences can be created bysubstituting one or more VH and/or VL CDR region sequences withstructurally similar sequences from the CDR sequences shown herein formonoclonal antibodies of the present disclosure.

Accordingly, the present disclosure provides an isolated monoclonalantibody or antigen binding region thereof comprising a heavy chain CDR1comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 3, 22, 40, 58, 76, 94, 112 and 130; a heavy chain CDR2comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 4, 23, 41, 59, 77, 95, 113 and 131; a heavy chain CDR3comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 5, 24, 42, 60, 78, 96, 114 and 132; a light chain CDR1comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 12, 31, 49, 67, 85, 103, 121 and 139; a light chain CDR2comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 13, 32, 50, 68, 86, 104, 122 and 140; and a light chain CDR3comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 14, 33, 51, 69, 87, 105, 123 and 141; wherein the antibodyspecifically binds cKIT.

In a specific aspect, an antibody or antibody fragment (e.g., antigenbinding fragments) that specifically binds to cKIT comprising a heavychain CDR1 of SEQ ID NO:3, a heavy chain CDR2 of SEQ ID NO: 4; a heavychain CDR3 of SEQ ID NO:5; a light chain CDR1 of SEQ ID NO:12; a lightchain CDR2 of SEQ ID NO: 13; and a light chain CDR3 of SEQ ID NO: 14.

In a specific aspect, an antibody or antibody fragment (e.g., antigenbinding fragments) that specifically binds to cKIT comprising a heavychain CDR1 of SEQ ID NO:22, a heavy chain CDR2 of SEQ ID NO: 23; a heavychain CDR3 of SEQ ID NO:24; a light chain CDR1 of SEQ ID NO:31; a lightchain CDR2 of SEQ ID NO: 32; and a light chain CDR3 of SEQ ID NO: 33.

In a specific aspect, an antibody or antibody fragment (e.g., antigenbinding fragments) that specifically binds to cKIT comprising a heavychain CDR1 of SEQ ID NO:40, a heavy chain CDR2 of SEQ ID NO: 41; a heavychain CDR3 of SEQ ID NO:42; a light chain CDR1 of SEQ ID NO:49; a lightchain CDR2 of SEQ ID NO: 50; and a light chain CDR3 of SEQ ID NO: 51.

In a specific aspect, an antibody or antibody fragment (e.g., antigenbinding fragments) that specifically binds to cKIT comprising a heavychain CDR1 of SEQ ID NO:58, a heavy chain CDR2 of SEQ ID NO: 59; a heavychain CDR3 of SEQ ID NO:60; a light chain CDR1 of SEQ ID NO:67; a lightchain CDR2 of SEQ ID NO: 68; and a light chain CDR3 of SEQ ID NO: 69.

In a specific aspect, an antibody or antibody fragment (e.g., antigenbinding fragments) that specifically binds to cKIT comprising a heavychain CDR1 of SEQ ID NO:76, a heavy chain CDR2 of SEQ ID NO: 77; a heavychain CDR3 of SEQ ID NO:78; a light chain CDR1 of SEQ ID NO:85; a lightchain CDR2 of SEQ ID NO: 86; and a light chain CDR3 of SEQ ID NO: 87.

In a specific aspect, an antibody or antibody fragment (e.g., antigenbinding fragments) that specifically binds to cKIT comprising a heavychain CDR1 of SEQ ID NO:94, a heavy chain CDR2 of SEQ ID NO: 95; a heavychain CDR3 of SEQ ID NO:96; a light chain CDR1 of SEQ ID NO:103; a lightchain CDR2 of SEQ ID NO: 104; and a light chain CDR3 of SEQ ID NO: 105.

In a specific aspect, an antibody or antibody fragment (e.g., antigenbinding fragments) that specifically binds to cKIT comprising a heavychain CDR1 of SEQ ID NO:112, a heavy chain CDR2 of SEQ ID NO: 113; aheavy chain CDR3 of SEQ ID NO:114; a light chain CDR1 of SEQ ID NO:121;a light chain CDR2 of SEQ ID NO: 122; and a light chain CDR3 of SEQ IDNO: 123.

In a specific aspect, an antibody or antibody fragment (e.g., antigenbinding fragments) that specifically binds to cKIT comprising a heavychain CDR1 of SEQ ID NO:130, a heavy chain CDR2 of SEQ ID NO: 131; aheavy chain CDR3 of SEQ ID NO:132; a light chain CDR1 of SEQ ID NO:139;a light chain CDR2 of SEQ ID NO: 140; and a light chain CDR3 of SEQ IDNO: 141.

In certain aspects, an antibody that specifically binds to cKIT is anantibody or antibody fragment (e.g., antigen binding fragment) that isdescribed in Table 1.

1. Identification of Epitopes and Antibodies that Bind to the SameEpitope

The present disclosure provides antibodies and antibody fragments (e.g.,antigen binding fragments) that bind to an epitope of within theextracellular domain of the cKIT receptor. In certain aspects theantibodies and antibody fragments can bind to epitopes with domains 1-3of the cKIT extracellular domain.

The present disclosure also provides antibodies and antibody fragments(e.g., antigen binding fragments) that bind to the same epitope as dothe anti-cKIT antibodies described in Table 1. Additional antibodies andantibody fragments (e.g., antigen binding fragments) can therefore beidentified based on their ability to cross-compete (e.g., tocompetitively inhibit the binding of, in a statistically significantmanner) with other antibodies in cKIT binding assays. The ability of atest antibody to inhibit the binding of antibodies and antibodyfragments (e.g., antigen binding fragments) of the present disclosure toa cKIT protein (e.g., human cKIT) demonstrates that the test antibodycan compete with that antibody or antibody fragment (e.g., antigenbinding fragments) for binding to cKIT; such an antibody may, accordingto non-limiting theory, bind to the same or a related (e.g., astructurally similar or spatially proximal) epitope on the cKIT proteinas the antibody or antibody fragment (e.g., antigen binding fragments)with which it competes. In a certain aspect, the antibody that binds tothe same epitope on cKIT as the antibodies or antibody fragments (e.g.,antigen binding fragments) of the present disclosure is a human orhumanized monoclonal antibody. Such human or humanized monoclonalantibodies can be prepared and isolated as described herein.

2. Further Alteration of the Framework of Fc Region

The present disclosure provides site-specific labeled immunoconjugates.These immunoconjugates can comprise modified antibodies or antigenbinding fragments thereof that further comprise modifications toframework residues within VH and/or VL, e.g. to improve the propertiesof the antibody. Typically such framework modifications are made todecrease the immunogenicity of the antibody. For example, one approachis to “back-mutate” one or more framework residues to the correspondinggermline sequence. More specifically, an antibody that has undergonesomatic mutation may contain framework residues that differ from thegermline sequence from which the antibody is derived. Such residues canbe identified by comparing the antibody framework sequences to thegermline sequences from which the antibody is derived. To return theframework region sequences to their germline configuration, the somaticmutations can be “back-mutated” to the germline sequence by, forexample, site-directed mutagenesis. Such “back-mutated” antibodies arealso intended to be encompassed.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T-cell epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 2003/0153043 by Carr et al.

In addition or alternative to modifications made within the framework orCDR regions, antibodies can be engineered to include modificationswithin the Fc region, typically to alter one or more functionalproperties of the antibody, such as serum half-life, complementfixation, Fc receptor binding, and/or antigen-dependent cellularcytotoxicity. Furthermore, an antibody can be chemically modified (e.g.,one or more chemical moieties can be attached to the antibody) or bemodified to alter its glycosylation, again to alter one or morefunctional properties of the antibody. Each of these aspects isdescribed in further detail below.

In one aspect, the hinge region of CH1 is modified such that the numberof cysteine residues in the hinge region is altered, e.g., increased ordecreased. This approach is described further in U.S. Pat. No. 5,677,425by Bodmer et al. The number of cysteine residues in the hinge region ofCH1 is altered to, for example, facilitate assembly of the light andheavy chains or to increase or decrease the stability of the antibody.

In another aspect, the Fc hinge region of an antibody is mutated todecrease the biological half-life of the antibody. More specifically,one or more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745 by Ward et al.

In yet other aspects, the Fc region is altered by replacing at least oneamino acid residue with a different amino acid residue to alter theeffector functions of the antibody. For example, one or more amino acidscan be replaced with a different amino acid residue such that theantibody has an altered affinity for an effector ligand but retains theantigen-binding ability of the parent antibody. The effector ligand towhich affinity is altered can be, for example, an Fc receptor or the C1component of complement. This approach is described in, e.g., U.S. Pat.Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another aspect, one or more amino acids selected from amino acidresidues can be replaced with a different amino acid residue such thatthe antibody has altered C1q binding and/or reduced or abolishedcomplement dependent cytotoxicity (CDC). This approach is described in,e.g., U.S. Pat. No. 6,194,551 by Idusogie et al.

In another aspect, one or more amino acid residues are altered tothereby alter the ability of the antibody to fix complement. Thisapproach is described in, e.g., the PCT Publication WO 94/29351 byBodmer et al. In a specific aspect, one or more amino acids of anantibody or antigen binding fragment thereof of the present disclosureare replaced by one or more allotypic amino acid residues, such as thoseshown in FIG. 4 for the IgG1 subclass and the kappa isotype. Allotypicamino acid residues also include, but are not limited to, the constantregion of the heavy chain of the IgG1, IgG2, and IgG3 subclasses as wellas the constant region of the light chain of the kappa isotype asdescribed by Jefferis et al., MAbs. 1:332-338 (2009).

In yet another aspect, the Fc region is modified to increase the abilityof the antibody to mediate antibody dependent cellular cytotoxicity(ADCC) and/or to increase the affinity of the antibody for an Fcγreceptor by modifying one or more amino acids. This approach isdescribed in, e.g., the PCT Publication WO 00/42072 by Presta. Moreover,the binding sites on human IgG1 for FcγR1, FcγRII, FcγRIII and FcRn havebeen mapped and variants with improved binding have been described (seeShields et al., J. Biol. Chem. 276:6591-6604, 2001).

In still another aspect, the glycosylation of an antibody is modified.For example, an aglycosylated antibody can be made (i.e., the antibodylacks glycosylation). Glycosylation can be altered to, for example,increase the affinity of the antibody for “antigen.” Such carbohydratemodifications can be accomplished by, for example, altering one or moresites of glycosylation within the antibody sequence. For example, one ormore amino acid substitutions can be made that result in elimination ofone or more variable region framework glycosylation sites to therebyeliminate glycosylation at that site. Such aglycosylation may increasethe affinity of the antibody for antigen. Such an approach is describedin, e.g., U.S. Pat. Nos. 5,714,350 and 6,350,861 by Co et al.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies to thereby produce an antibody with alteredglycosylation. For example, EP 1,176,195 by Hang et al. describes a cellline with a functionally disrupted FUT8 gene, which encodes a fucosyltransferase, such that antibodies expressed in such a cell line exhibithypofucosylation. PCT Publication WO 03/035835 by Presta describes avariant CHO cell line, Lecl3 cells, with reduced ability to attachfucose to Asn(297)-linked carbohydrates, also resulting inhypofucosylation of antibodies expressed in that host cell (see alsoShields et al., (2002) J. Biol. Chem. 277:26733-26740). PCT PublicationWO 99/54342 by Umana et al. describes cell lines engineered to expressglycoprotein-modifying glycosyl transferases (e.g., beta(1,4)-Nacetylglucosaminyltransferase III (GnTIII)) such that antibodiesexpressed in the engineered cell lines exhibit increased bisectingGlcNac structures which results in increased ADCC activity of theantibodies (see also Umana et al., Nat. Biotech. 17:176-180, 1999).

In another aspect, the antibody is modified to increase its biologicalhalf-life. Various approaches are possible. For example, one or more ofthe following mutations can be introduced: T252L, T254S, T256F, asdescribed in U.S. Pat. No. 6,277,375 to Ward. Alternatively, to increasethe biological half-life, the antibody can be altered within the CH1 orCL region to contain a salvage receptor binding epitope taken from twoloops of a CH2 domain of an Fc region of an IgG, as described in U.S.Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.

3. Production of the cKIT Antibodies

Anti-cKIT antibodies and antibody fragments (e.g., antigen bindingfragments) thereof can be produced by any means known in the art,including but not limited to, recombinant expression, chemicalsynthesis, and enzymatic digestion of antibody tetramers, whereasfull-length monoclonal antibodies can be obtained by, e.g., hybridoma orrecombinant production. Recombinant expression can be from anyappropriate host cells known in the art, for example, mammalian hostcells, bacterial host cells, yeast host cells, insect host cells, etc.

The disclosure further provides polynucleotides encoding the antibodiesdescribed herein, e.g., polynucleotides encoding heavy or light chainvariable regions or segments comprising the complementarity determiningregions as described herein. In some aspects, the polynucleotideencoding the heavy chain variable regions has at least 85%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acidsequence identity with a polynucleotide selected from the groupconsisting of SEQ ID NOs: 30, 48, 66, 84, 102, 120 and 137. In someaspects, the polynucleotide encoding the light chain variable regionshas at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% nucleic acid sequence identity with a polynucleotide selectedfrom the group consisting of SEQ ID NOs:39, 57, 75, 93, 111, 129 and147.

In some aspects, the polynucleotide encoding the heavy chain has atleast 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO:30, 48, 66, 84, 102, 120. In some aspects, the polynucleotide encodingthe light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with apolynucleotide of SEQ ID NO: 39, 57, 75, 93, 111, 129 and 147.

The polynucleotides of the present disclosure can encode only thevariable region sequence of an anti-cKIT antibody. They can also encodeboth a variable region and a constant region of the antibody. Some ofthe polynucleotide sequences encode a polypeptide that comprisesvariable regions of both the heavy chain and the light chain of one ofan exemplified anti-cKIT antibody. Some other polynucleotides encode twopolypeptide segments that respectively are substantially identical tothe variable regions of the heavy chain and the light chain of one ofthe mouse antibodies.

The polynucleotide sequences can be produced by de novo solid-phase DNAsynthesis or by PCR mutagenesis of an existing sequence (e.g., sequencesas described in the Examples below) encoding an anti-cKIT antibody orits binding fragment. Direct chemical synthesis of nucleic acids can beaccomplished by methods known in the art, such as the phosphotriestermethod of Narang et al., Meth. Enzymol. 68:90, 1979; the phosphodiestermethod of Brown et al., Meth. Enzymol. 68:109, 1979; thediethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22:1859,1981; and the solid support method of U.S. Pat. No. 4,458,066.Introducing mutations to a polynucleotide sequence by PCR can beperformed as described in, e.g., PCR Technology: Principles andApplications for DNA Amplification, H.A. Erlich (Ed.), Freeman Press,NY, N.Y., 1992; PCR Protocols: A Guide to Methods and Applications,Innis et al. (Ed.), Academic Press, San Diego, Calif., 1990; Mattila etal., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods andApplications 1:17, 1991.

Also provided in the present disclosure are expression vectors and hostcells for producing the anti-cKIT antibodies described above. Variousexpression vectors can be employed to express the polynucleotidesencoding the anti-cKIT antibody chains or binding fragments. Bothviral-based and nonviral expression vectors can be used to produce theantibodies in a mammalian host cell. Nonviral vectors and systemsinclude plasmids, episomal vectors, typically with an expressioncassette for expressing a protein or RNA, and human artificialchromosomes (see, e.g., Harrington et al., Nat Genet. 15:345, 1997). Forexample, nonviral vectors useful for expression of the anti-cKITpolynucleotides and polypeptides in mammalian (e.g., human) cellsinclude pThioHis A, B & C, pcDNA3.1/H is, pEBVHis A, B & C (Invitrogen,San Diego, Calif.), MPSV vectors, and numerous other vectors known inthe art for expressing other proteins. Useful viral vectors includevectors based on retroviruses, adenoviruses, adenoassociated viruses,herpes viruses, vectors based on SV40, papilloma virus, HBP Epstein Barrvirus, vaccinia virus vectors and Semliki Forest virus (SFV). See, Brentet al., supra; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeldet al., Cell 68:143, 1992.

The choice of expression vector depends on the intended host cells inwhich the vector is to be expressed. Typically, the expression vectorscontain a promoter and other regulatory sequences (e.g., enhancers) thatare operably linked to the polynucleotides encoding an anti-cKITantibody chain or fragment. In some aspects, an inducible promoter isemployed to prevent expression of inserted sequences except underinducing conditions. Inducible promoters include, e.g., arabinose, lacZ,metallothionein promoter or a heat shock promoter. Cultures oftransformed organisms can be expanded under noninducing conditionswithout biasing the population for coding sequences whose expressionproducts are better tolerated by the host cells. In addition topromoters, other regulatory elements may also be required or desired forefficient expression of an anti-cKIT antibody chain or fragment. Theseelements typically include an ATG initiation codon and adjacent ribosomebinding site or other sequences. In addition, the efficiency ofexpression may be enhanced by the inclusion of enhancers appropriate tothe cell system in use (see, e.g., Scharf et al., Results Probl. CellDiffer. 20:125, 1994; and Bittner et al., Meth. Enzymol., 153:516,1987). For example, the SV40 enhancer or CMV enhancer may be used toincrease expression in mammalian host cells.

The expression vectors may also provide a secretion signal sequenceposition to form a fusion protein with polypeptides encoded by insertedanti-cKIT antibody sequences. More often, the inserted anti-cKITantibody sequences are linked to a signal sequences before inclusion inthe vector. Vectors to be used to receive sequences encoding anti-cKITantibody light and heavy chain variable domains sometimes also encodeconstant regions or parts thereof. Such vectors allow expression of thevariable regions as fusion proteins with the constant regions therebyleading to production of intact antibodies or fragments thereof.Typically, such constant regions are human.

The host cells for harboring and expressing the anti-cKIT antibodychains can be either prokaryotic or eukaryotic. E. coli is oneprokaryotic host useful for cloning and expressing the polynucleotidesof the present disclosure. Other microbial hosts suitable for useinclude bacilli, such as Bacillus subtilis, and otherenterobacteriaceae, such as Salmonella, Serratia, and variousPseudomonas species. In these prokaryotic hosts, one can also makeexpression vectors, which typically contain expression control sequencescompatible with the host cell (e.g., an origin of replication). Inaddition, any number of a variety of well-known promoters will bepresent, such as the lactose promoter system, a tryptophan (trp)promoter system, a beta-lactamase promoter system, or a promoter systemfrom phage lambda. The promoters typically control expression,optionally with an operator sequence, and have ribosome binding sitesequences and the like, for initiating and completing transcription andtranslation. Other microbes, such as yeast, can also be employed toexpress anti-cKIT polypeptides. Insect cells in combination withbaculovirus vectors can also be used.

In other aspects, mammalian host cells are used to express and producethe anti-cKIT polypeptides of the present disclosure. For example, theycan be either a hybridoma cell line expressing endogenous immunoglobulingenes (e.g., the myeloma hybridoma clones as described in the Examples)or a mammalian cell line harboring an exogenous expression vector (e.g.,the SP2/0 myeloma cells exemplified below). These include any normalmortal or normal or abnormal immortal animal or human cell. For example,a number of suitable host cell lines capable of secreting intactimmunoglobulins have been developed, including the CHO cell lines,various COS cell lines, HeLa cells, myeloma cell lines, transformedB-cells and hybridomas. The use of mammalian tissue cell culture toexpress polypeptides is discussed generally in, e.g., Winnacker, FromGenes to Clones, VCH Publishers, N.Y., N.Y., 1987. Expression vectorsfor mammalian host cells can include expression control sequences, suchas an origin of replication, a promoter, and an enhancer (see, e.g.,Queen et al., Immunol. Rev. 89:49-68, 1986), and necessary processinginformation sites, such as ribosome binding sites, RNA splice sites,polyadenylation sites, and transcriptional terminator sequences. Theseexpression vectors usually contain promoters derived from mammaliangenes or from mammalian viruses. Suitable promoters may be constitutive,cell type-specific, stage-specific, and/or modulatable or regulatable.Useful promoters include, but are not limited to, the metallothioneinpromoter, the constitutive adenovirus major late promoter, thedexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIIIpromoter, the constitutive MPSV promoter, the tetracycline-inducible CMVpromoter (such as the human immediate-early CMV promoter), theconstitutive CMV promoter, and promoter-enhancer combinations known inthe art.

Methods for introducing expression vectors containing the polynucleotidesequences of interest vary depending on the type of cellular host. Forexample, calcium chloride transfection is commonly utilized forprokaryotic cells, whereas calcium phosphate treatment orelectroporation may be used for other cellular hosts (see generallySambrook et al., supra). Other methods include, e.g., electroporation,calcium phosphate treatment, liposome-mediated transformation, injectionand microinjection, ballistic methods, virosomes, immunoliposomes,polycation:nucleic acid conjugates, naked DNA, artificial virions,fusion to the herpes virus structural protein VP22 (Elliot and O'Hare,Cell 88:223, 1997), agent-enhanced uptake of DNA, and ex vivotransduction. For long-term, high-yield production of recombinantproteins, stable expression will often be desired. For example, celllines which stably express anti-cKIT antibody chains or bindingfragments can be prepared using expression vectors which contain viralorigins of replication or endogenous expression elements and aselectable marker gene. Following introduction of the vector, cells maybe allowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth of cellswhich successfully express the introduced sequences in selective media.Resistant, stably transfected cells can be proliferated using tissueculture techniques appropriate to the cell type.

Therapeutic and Diagnostic Uses

The antibodies, antibody fragments (e.g., antigen binding fragments),and antibody drug conjugates of the present disclosure are useful in avariety of applications including, but not limited to, treatment ofcancer, such as solid cancers. In certain aspects, the antibodies,antibody fragments (e.g., antigen binding fragments), and antibody drugconjugates are useful for inhibiting tumor growth, inducingdifferentiation, reducing tumor volume, and/or reducing thetumorigenicity of a tumor. The methods of use can be in vitro, ex vivo,or in vivo methods.

In one aspect, the antibodies, antibody fragments (e.g., antigen bindingfragments), and antibody drug conjugates are useful for detecting thepresence of cKIT in a biological sample. The term “detecting” as usedherein encompasses quantitative or qualitative detection. In certainaspects, a biological sample comprises a cell or tissue. In certainaspects, such tissues include normal and/or cancerous tissues thatexpress cKIT at higher levels relative to other tissues.

In one aspect, the present disclosure provides a method of detecting thepresence of cKIT in a biological sample. In certain aspects, the methodcomprises contacting the biological sample with an anti-cKIT antibodyunder conditions permissive for binding of the antibody to the antigen,and detecting whether a complex is formed between the antibody and theantigen.

Also included is a method of diagnosing a disorder associated withincreased expression of cKIT. In certain aspects, the method comprisescontacting a test cell with an anti-cKIT antibody; determining the levelof expression (either quantitatively or qualitatively) of cKIT on thetest cell by detecting binding of the anti-cKIT antibody to the cKITantigen; and comparing the level of expression of cKIT in the test cellwith the level of expression of cKIT in a control cell (e.g., a normalcell of the same tissue origin as the test cell or a cell that expressescKIT at levels comparable to such a normal cell), wherein a higher levelof expression of cKIT on the test cell as compared to the control cellindicates the presence of a disorder associated with increasedexpression of cKIT. In certain aspects, the test cell is obtained froman individual suspected of having a disorder associated with increasedexpression of cKIT. In certain aspects, the disorder is a cellproliferative disorder, such as a cancer or a tumor.

In certain aspects, a method of diagnosis or detection, such as thosedescribed above, comprises detecting binding of an anti-cKIT antibody tocKIT expressed on the surface of a cell or in a membrane preparationobtained from a cell expressing cKIT on its surface. An exemplary assayfor detecting binding of an anti-cKIT antibody to cKIT expressed on thesurface of a cell is a “FACS” assay.

Certain other methods can be used to detect binding of anti-cKITantibodies to cKIT. Such methods include, but are not limited to,antigen-binding assays that are well known in the art, such as westernblots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay),“sandwich” immunoassays, immunoprecipitation assays, fluorescentimmunoassays, protein A immunoassays, and immunohistochemistry (IHC).

In certain aspects, anti-cKIT antibodies are labeled. Labels include,but are not limited to, labels or moieties that are detected directly(such as fluorescent, chromophoric, electron-dense, chemiluminescent,and radioactive labels), as well as moieties, such as enzymes orligands, that are detected indirectly, e.g., through an enzymaticreaction or molecular interaction.

In certain aspects, anti-cKIT antibodies are immobilized on an insolublematrix. Immobilization entails separating the anti-cKIT antibody fromany cKIT proteins that remains free in solution. This conventionally isaccomplished by either insolubilizing the anti-cKIT antibody before theassay procedure, as by adsorption to a water-insoluble matrix or surface(Bennich et al, U.S. Pat. No. 3,720,760), or by covalent coupling (forexample, using glutaraldehyde cross-linking), or by insolubilizing theanti-cKIT antibody after formation of a complex between the anti-cKITantibody and cKIT protein, e.g., by immunoprecipitation.

Any of the above aspects of diagnosis or detection can be carried outusing an immunoconjugate of the present disclosure in place of or inaddition to an anti-cKIT antibody.

In one aspect, the disclosure provides for a method of treating,preventing or ameliorating a disease comprising administering theantibodies, antibody fragments (e.g., antigen binding fragments), andantibody drug conjugates to a patient, thereby treating the disease. Incertain aspects, the disease treated with the antibodies, antibodyfragments (e.g., antigen binding fragments), and antibody drugconjugates is a cancer. Examples of diseases which can be treated and/orprevented include, but are not limited to, gastrointestinal stromaltumors (GIST), small cell lung cancer (SCLC), acute myelod leukemia(AML), melanoma, mast cell leukemia (MCL), mastocytosis,neurofibromatosis, breast cancer, non-small cell lung cancer (NSCLC),and pancreatic cancer. In certain aspects, the cancer is characterizedby cKIT expressing cells to which the antibodies, antibody fragments(e.g., antigen binding fragments), and antibody drug conjugates canspecifically bind.

The present disclosure provides for methods of treating cancercomprising administering a therapeutically effective amount of theantibodies, antibody fragments (e.g., antigen binding fragments), orantibody drug conjugates. In certain aspects, the cancer is a solidcancer. In certain aspects, the subject is a human.

In certain aspects, the method of inhibiting tumor growth comprisesadministering to a subject a therapeutically effective amount of theantibodies, antibody fragments (e.g., antigen binding fragments), orantibody drug conjugates. In certain aspects, the subject is a human. Incertain aspects, the subject has a tumor or has had a tumor removed.

In certain aspects, the tumor expresses the cKIT to which the anti-cKITantibody binds. In certain aspects, the tumor overexpresses the humancKIT.

For the treatment of the disease, the appropriate dosage of theantibodies, antibody fragments (e.g., antigen binding fragments), orantibody drug conjugates depend on various factors, such as the type ofdisease to be treated, the severity and course of the disease, theresponsiveness of the disease, previous therapy, patient's clinicalhistory, and so on. The antibody or agent can be administered one timeor over a series of treatments lasting from several days to severalmonths, or until a cure is effected or a diminution of the disease stateis achieved (e.g., reduction in tumor size). Optimal dosing schedulescan be calculated from measurements of drug accumulation in the body ofthe patient and will vary depending on the relative potency of anindividual antibody, antibody fragment (e.g., antigen binding fragment),or antibody drug conjugates. In certain aspects, dosage is from 0.01 mgto 10 mg (e.g., 0.01 mg, 0.05 mg, 0.1 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4mg, 5 mg, 7 mg, 8 mg, 9 mg, or 10 mg) per kg of body weight, and can begiven once or more daily, weekly, monthly or yearly. In certain aspects,the antibody, antibody fragment (e.g., antigen binding fragment), orantibody drug conjugate of the present disclosure is given once everytwo weeks or once every three weeks. The treating physician can estimaterepetition rates for dosing based on measured residence times andconcentrations of the drug in bodily fluids or tissues.

Combination Therapy

In certain instances, an antibody, antibody fragment (e.g., antigenbinding fragment), or antibody drug conjugate of the present disclosureis combined with other therapeutic agents, such as other anti-canceragents, anti-allergic agents, anti-nausea agents (or anti-emetics), painrelievers, cytoprotective agents, and combinations thereof.

General Chemotherapeutic agents considered for use in combinationtherapies include anastrozole (Arimidex®), bicalutamide (Casodex®),bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection(Busulfex®), capecitabine (Xeloda®),N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®),carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®),cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®),cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposomeinjection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin(Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®),daunorubicin citrate liposome injection (DaunoXome®), dexamethasone,docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®),etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil(Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, Gemcitabine(difluorodeoxycitidine), hydroxyurea (Hydrea®), Idarubicin (Idamycin®),ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®),leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine(Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®),mylotarg, paclitaxel (Taxol®), phoenix (Yttrium90/MX-DTPA), pentostatin,polifeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate(Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine(Tirazone®), topotecan hydrochloride for injection (Hycamptin®),vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine(Navelbine®).

In one aspect, an antibody, antibody fragment (e.g., antigen bindingfragment), or antibody drug conjugate of the present disclosure iscombined in a pharmaceutical combination formulation, or dosing regimenas combination therapy, with a second compound having anti-cancerproperties. The second compound of the pharmaceutical combinationformulation or dosing regimen can have complementary activities to theantibody or immunoconjugate of the combination such that they do notadversely affect each other. For example, an antibody, antibody fragment(e.g., antigen binding fragment), or antibody drug conjugate of thepresent disclosure can be administered in combination with, but notlimited to, a chemotherapeutic agent, a tyrosine kinase inhibitor, forexample, Imatinib, and other cKIT pathway inhibitors.

The term “pharmaceutical combination” as used herein refers to either afixed combination in one dosage unit form, or non-fixed combination or akit of parts for the combined administration where two or moretherapeutic agents may be administered independently at the same time orseparately within time intervals, especially where these time intervalsallow that the combination partners show a cooperative, e.g. synergisticeffect.

The term “combination therapy” refers to the administration of two ormore therapeutic agents to treat a therapeutic condition or disorderdescribed in the present disclosure. Such administration encompassesco-administration of these therapeutic agents in a substantiallysimultaneous manner, such as in a single capsule having a fixed ratio ofactive ingredients. Alternatively, such administration encompassesco-administration in multiple, or in separate containers (e.g.,capsules, powders, and liquids) for each active ingredient. Powdersand/or liquids may be reconstituted or diluted to a desired dose priorto administration. In addition, such administration also encompasses useof each type of therapeutic agent in a sequential manner, either atapproximately the same time or at different times. In either case, thetreatment regimen will provide beneficial effects of the drugcombination in treating the conditions or disorders described herein.

The combination therapy can provide “synergy” and prove “synergistic”,i.e., the effect achieved when the active ingredients used together isgreater than the sum of the effects that results from using thecompounds separately. A synergistic effect can be attained when theactive ingredients are: (1) co-formulated and administered or deliveredsimultaneously in a combined, unit dosage formulation; (2) delivered byalternation or in parallel as separate formulations; or (3) by someother regimen. When delivered in alternation therapy, a synergisticeffect can be attained when the compounds are administered or deliveredsequentially, e.g., by different injections in separate syringes. Ingeneral, during alternation therapy, an effective dosage of each activeingredient is administered sequentially, i.e., serially, whereas incombination therapy, effective dosages of two or more active ingredientsare administered together.

In one aspect, the present disclosure provides a method of treatingcancer by administering to a subject in need thereof an antibody drugconjugate in combination with one or more tyrosine kinase inhibitors,including but not limited to, EGFR inhibitors, Her2 inhibitors, Her3inhibitors, IGFR inhibitors, and Met inhibitors.

For example, tyrosine kinase inhibitors include but are not limited to,Erlotinib hydrochloride (Tarceva®); Linifanib(N-[4-(3-amino-1H-indazol-4-yl)phenyl]-N′-(2-fluoro-5-methylphenyl)urea,also known as ABT 869, available from Genentech); Sunitinib malate(Sutent®); Bosutinib(4-[(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[3-(4-methylpiperazin-1-yl)propoxy]quinoline-3-carbonitrile,also known as SKI-606, and described in U.S. Pat. No. 6,780,996);Dasatinib (Sprycel®); Pazopanib (Votrient®); Sorafenib (Nexavar®);Zactima (ZD6474); nilotinib (Tasigna®); Regorafenib (Stivarga®) andImatinib or Imatinib mesylate (Gilvec® and Gleevec®).

Epidermal growth factor receptor (EGFR) inhibitors include but are notlimited to, Erlotinib hydrochloride (Tarceva®), Gefitnib (Iressa®);N-[4-[(3-Chloro-4-fluorophenyl)amino]-7-[[(3″S″)-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4(dimethylamino)-2-butenamide,Tovok®); Vandetanib (Caprelsa®); Lapatinib (Tykerb®);(3R,4R)-4-Amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)piperidin-3-ol(BMS690514); Canertinib dihydrochloride (CI-1033);6-[4-[(4-Ethyl-1-piperazinyl)methyl]phenyl]-N-[(1R)-1-phenylethyl]-7H-Pyrrolo[2,3-d]pyrimidin-4-amine(AEE788, CAS 497839-62-0); Mubritinib (TAK165); Pelitinib (EKB569);Afatinib (BIBW2992); Neratinib (HKI-272);N-[4-[[1-[(3-Fluorophenyl)methyl]-1H-indazol-5-yl]amino]-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yl]-carbamicacid, (3S)-3-morpholinylmethyl ester (BMS599626);N-(3,4-Dichloro-2-fluorophenyl)-6-methoxy-7-[[(3aα,5β,6aα)-octahydro-2-methylcyclopenta[c]pyrrol-5-yl]methoxy]-4-quinazolinamine(XL647, CAS 781613-23-8); and4-[4-[[(1R)-1-Phenylethyl]amino]-7H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol(PKI166, CAS187724-61-4).

EGFR antibodies include but are not limited to, Cetuximab (Erbitux®);Panitumumab (Vectibix®); Matuzumab (EMD-72000); Trastuzumab(Herceptin®); Nimotuzumab (hR3); Zalutumumab; TheraCIM h-R3; MDX0447(CAS 339151-96-1); and ch806 (mAb-806, CAS 946414-09-1).

Human Epidermal Growth Factor Receptor 2 (HER2 receptor) (also known asNeu, ErbB-2, CD340, or p185) inhibitors include but are not limited to,Trastuzumab (Herceptin®); Pertuzumab (Omnitarg®); Neratinib (HKI-272,(2E)-N-[4-[[3-chloro-4-[(pyridin-2-yl)methoxy]phenyl]amino]-3-cyano-7-ethoxyquinolin-6-yl]-4-(dimethylamino)but-2-enamide,and described PCT Publication No. WO 05/028443); Lapatinib or Lapatinibditosylate (Tykerb®);(3R,4R)-4-amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)piperidin-3-ol(BMS690514);(2E)-N-[4-[(3-Chloro-4-fluorophenyl)amino]-7-[[(3S)-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4-(dimethylamino)-2-butenamide(BIBW-2992, CAS 850140-72-6);N-[4-[[1-[(3-Fluorophenyl)methyl]-1H-indazol-5-yl]amino]-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yl]-carbamicacid, (3S)-3-morpholinylmethyl ester (BMS 599626, CAS 714971-09-2);Canertinib dihydrochloride (PD183805 or CI-1033); andN-(3,4-Dichloro-2-fluorophenyl)-6-methoxy-7-[[(3aα,5β,6aα)-octahydro-2-methylcyclopenta[c]pyrrol-5-yl]methoxy]-4-quinazolinamine(XL647, CAS 781613-23-8).

HER3 inhibitors include but are not limited to, LJM716, MM-121, AMG-888,RG7116, REGN-1400, AV-203, MP-RM-1, MM-111, and MEHD-7945A.

MET inhibitors include but are not limited to, Cabozantinib (XL184, CAS849217-68-1); Foretinib (GSK1363089, formerly XL880, CAS 849217-64-7);Tivantinib (ARQ197, CAS 1000873-98-2);1-(2-Hydroxy-2-methylpropyl)-N-(5-(7-methoxyquinolin-4-yloxy)pyridin-2-yl)-5-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide(AMG 458); Cryzotinib (Xalkori®, PF-02341066);(3Z)-5-(2,3-Dihydro-1H-indol-1-ylsulfonyl)-3-({3,5-dimethyl-4-[(4-methylpiperazin-1-yl)carbonyl]-1H-pyrrol-2-yl}methylene)-1,3-dihydro-2H-indol-2-one(SU11271);(3Z)—N-(3-Chlorophenyl)-3-({3,5-dimethyl-4-[(4-methylpiperazin-1-yl)carbonyl]-1H-pyrrol-2-yl}methylene)-N-methyl-2-oxoindoline-5-sulfonamide(SU11274);(3Z)—N-(3-Chlorophenyl)-3-{[3,5-dimethyl-4-(3-morpholin-4-ylpropyl)-1H-pyrrol-2-yl]methylene}-N-methyl-2-oxoindoline-5-sulfonamide(SU11606);6-[Difluoro[6-(1-methyl-1H-pyrazol-4-yl)-1,2,4-triazolo[4,3-b]pyridazin-3-yl]methyl]-quinoline(JNJ38877605, CAS 943540-75-8);2-[4-[1-(Quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-6-yl]-1H-pyrazol-1-yl]ethanol(PF04217903, CAS 956905-27-4);N-((2R)-1,4-Dioxan-2-ylmethyl)-N-methyl-N′-[3-(1-methyl-1H-pyrazol-4-yl)-5-oxo-5H-benzo[4,5]cyclohepta[1,2-b]pyridin-7-yl]sulfamide(MK2461, CAS 917879-39-1);6-[[6-(1-Methyl-1H-pyrazol-4-yl)-1,2,4-triazolo[4,3-b]pyridazin-3-yl]thio]-quinoline(SGX523, CAS1022150-57-7); and(3Z)-5-[[(2,6-Dichlorophenyl)methyl]sulfonyl]-3-[[3,5-dimethyl-4-[[(2R)-2-(1-pyrrolidinylmethyl)-1-pyrrolidinyl]carbonyl]-1H-pyrrol-2-yl]methylene]-1,3-dihydro-2H-indol-2-one(PHA665752, CAS 477575-56-7).

IGF1R inhibitors include but are not limited to, BMS-754807, XL-228,OSI-906, GSK0904529A, A-928605, AXL1717, KW-2450, MK0646, AMG479,IMCA12, MEDI-573, and BI836845. See e.g., Yee, JNCI, 104; 975 (2012) forreview.

In another aspect, the present disclosure provides a method of treatingcancer by administering to a subject in need thereof an antibody drugconjugate in combination with one or more FGF downstream signalingpathway inhibitors, including but not limited to, MEK inhibitors, Brafinhibitors, PI3K/Akt inhibitors, SHP2 inhibitors, and also mTor.

For example, mitogen-activated protein kinase (MEK) inhibitors includebut are not limited to, XL-518 (also known as GDC-0973, Cas No.1029872-29-4, available from ACC Corp.);2-[(2-Chloro-4-iodophenyl)amino]-N-(cyclopropylmethoxy)-3,4-difluoro-benzamide(also known as CI-1040 or PD184352 and described in PCT Publication No.WO2000035436);N-[(2R)-2,3-Dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide(also known as PD0325901 and described in PCT Publication No.WO2002006213);2,3-Bis[amino[(2-aminophenyl)thio]methylene]-butanedinitrile (also knownas U0126 and described in U.S. Pat. No. 2,779,780);N-[3,4-Difluoro-2-[(2-fluoro-4-iodophenyl)amino]-6-methoxyphenyl]-1-[(2R)-2,3-dihydroxypropyl]-cyclopropanesulfonamide(also known as RDEA119 or BAY869766 and described in PCT Publication No.WO2007014011);(3S,4R,5Z,8S,9S,11E)-[4-(Ethylamino)-8,9,16-trihydroxy-3,4-dimethyl-3,4,9,19-tetrahydro-1H-2-benzoxacyclotetradecine-1,7(8H)-dione](also known as E6201 and described in PCT Publication No. WO2003076424);2′-Amino-3′-methoxyflavone (also known as PD98059 available from BiaffinGmbH & Co., KG, Germany); Vemurafenib (PLX-4032, CAS 918504-65-1);(R)-3-(2,3-Dihydroxypropyl)-6-fluoro-5-(2-fluoro-4-iodophenylamino)-8-methylpyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione(TAK-733, CAS1035555-63-5); Pimasertib (AS-703026, CAS1204531-26-9); andTrametinib dimethyl sulfoxide (GSK-1120212, CAS1204531-25-80).

Phosphoinositide 3-kinase (PI3K) inhibitors include but are not limitedto,4-[2-(1H-Indazol-4-yl)-6-[[4-(methylsulfonyl)piperazin-1-yl]methyl]thieno[3,2-d]pyrimidin-4-yl]morpholine(also known as GDC 0941 and described in PCT Publication Nos. WO09/036,082 and WO 09/055,730);2-Methyl-2-[4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydroimidazo[4,5-c]quinolin-1-yl]phenyl]propionitrile(also known as BEZ 235 or NVP-BEZ 235, and described in PCT PublicationNo. WO 06/122806);4-(trifluoromethyl)-5-(2,6-dimorpholinopyrimidin-4-yl)pyridin-2-amine(also known as BKM120 or NVP-BKM120, and described in PCT PublicationNo. WO2007/084786); Tozasertib (VX680 or MK-0457, CAS 639089-54-6);(5Z)-5-[[4-(4-Pyridinyl)-6-quinolinyl]methylene]-2,4-thiazolidinedione(GSK1059615, CAS 958852-01-2);(1E,4S,4aR,5R,6aS,9aR)-5-(Acetyloxy)-1-[(di-2-propenylamino)methylene]-4,4-a,5,6,6a,8,9,9a-octahydro-11-hydroxy-4-(methoxymethyl)-4-a,6a-dimethyl-cyclopenta[5,6]naphtho[1,2-c]pyran-2,7,10(1H)-trione(PX866, CAS 502632-66-8); and 8-Phenyl-2-(morpholin-4-yl)-chromen-4-one(LY294002, CAS154447-36-6).

mTor include but are not limited to, Temsirolimus (Torisel®);Ridaforolimus (formally known as deferolimus,(1R,2R,4S)-4-[(2R)-2[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0^(4,9)]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinate, also known as AP23573 and MK8669, and described inPCT Publication No. WO 03/064383); Everolimus (Afinitor® or RAD001);Rapamycin (AY22989, Sirolimus®); Simapimod (CAS164301-51-3);(5-{2,4-Bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol(AZD8055);2-Amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one(PF04691502, CAS1013101-36-4); andN²-[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartylL-serine-,inner salt (SF1126, CAS 936487-67-1).

In yet another aspect, the present disclosure provides a method oftreating cancer by administering to a subject in need thereof anantibody drug conjugate in combination with one or more pro-apoptotics,including but not limited to, IAP inhibitors, Bcl2 inhibitors, MCl1inhibitors, Trail agents, Chk inhibitors.

For examples, IAP inhibitors include but are not limited to, LCL161,GDC-0917, AEG-35156, AT406, and TL32711. Other examples of IAPinhibitors include but are not limited to those disclosed inWO04/005284, WO 04/007529, WO05/097791, WO 05/069894, WO 05/069888, WO05/094818, US2006/0014700, US2006/0025347, WO 06/069063, WO 06/010118,WO 06/017295, and WO08/134,679, all of which are incorporated herein byreference.

BCL-2 inhibitors include but are not limited to,4-[4-[[2-(4-Chlorophenyl)-5,5-dimethyl-1-cyclohexen-1-yl]methyl]-1-piperazinyl]-N-[[4-[[(1R)-3-(4-morpholinyl)-1-[(phenylthio)methyl]propyl]amino]-3-[(trifluoromethyl)sulfonyl]phenyl]sulfonyl]benzamide(also known as ABT-263 and described in PCT Publication No. WO09/155,386); Tetrocarcin A; Antimycin; Gossypol ((−)BL-193); Obatoclax;Ethyl-2-amino-6-cyclopentyl-4-(1-cyano-2-ethoxy-2-oxoethyl)-4Hchromone-3-carboxylate(HA14-1); Oblimersen (G3139, Genasense®); Bak BH3 peptide; (−)-Gossypolacetic acid (AT-101);4-[4-[(4′-Chloro[1,1′-biphenyl]-2-yl)methyl]-1-piperazinyl]-N-[[4-[[(1R)-3-(dimethylamino)-1-[(phenylthio)methyl]propyl]amino]-3-nitrophenyl]sulfonyl]-benzamide(ABT-737, CAS 852808-04-9); and Navitoclax (ABT-263, CAS 923564-51-6).

Proapoptotic receptor agonists (PARAs) including DR4 (TRAILR1) and DR5(TRAILR2), including but are not limited to, Dulanermin (AMG-951,RhApo2L/TRAIL); Mapatumumab (HRS-ETR1, CAS 658052-09-6); Lexatumumab(HGS-ETR2, CAS 845816-02-6); Apomab (Apomab®); Conatumumab (AMG655, CAS896731-82-1); and Tigatuzumab (CS1008, CAS 946415-34-5, available fromDaiichi Sankyo).

Checkpoint Kinase (CHK) inhibitors include but are not limited to,7-Hydroxystaurosporine (UCN-01);6-Bromo-3-(1-methyl-1H-pyrazol-4-yl)-5-(3R)-3-piperidinyl-pyrazolo[1,5-a]pyrimidin-7-amine(SCH900776, CAS 891494-63-6);5-(3-Fluorophenyl)-3-ureidothiophene-2-carboxylic acidN—[(S)-piperidin-3-yl]amide (AZD7762, CAS 860352-01-8);4-[((3S)-1-Azabicyclo[2.2.2]oct-3-yl)amino]-3-(1H-benzimidazol-2-yl)-6-chloroquinolin-2(1H)-one(CHIR 124, CAS 405168-58-3); 7-Aminodactinomycin (7-AAD),Isogranulatimide, debromohymenialdisine;N-[5-Bromo-4-methyl-2-[(2S)-2-morpholinylmethoxy]-phenyl]-N′-(5-methyl-2-pyrazinyl)urea(LY2603618, CAS 911222-45-2); Sulforaphane (CAS4478-93-7,4-Methylsulfinylbutyl isothiocyanate);9,10,11,12-Tetrahydro-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocine-1,3(2H)-dione(SB-218078, CAS135897-06-2); and TAT-S216A (Sha et al., Mol. Cancer.Ther 2007; 6(1):147-153), and CBP501((d-Bpa)sws(d-Phe-F5)(d-Cha)rrrqrr).

In one aspect, the present disclosure provides a method of treatingcancer by administering to a subject in need thereof an antibody drugconjugate in combination with one or more FGFR inhibitors. For example,FGFR inhibitors include but are not limited to, Brivanib alaninate(BMS-582664,(S)-((R)-1-(4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy)propan-2-yl)-2-aminopropanoate);Vargatef (BIBF1120, CAS 928326-83-4); Dovitinib dilactic acid (TKI258,CAS 852433-84-2);3-(2,6-Dichloro-3,5-dimethoxy-phenyl)-1-{6-[4-(4-ethyl-piperazin-1-yl)-phenylamino]-pyrimidin-4-yl}-1-methyl-urea(BGJ398, CAS 872511-34-7); Danusertib (PHA-739358); and (PD173074, CAS219580-11-7). In a specific aspect, the present disclosure provides amethod of treating cancer by administering to a subject in need thereofan antibody drug conjugate in combination with an FGFR2 inhibitor, suchas3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-(6((4-(4-ethylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)-1-methylurea(also known as BGJ-398); or4-amino-5-fluoro-3-(5-(4-methylpiperazin1-yl)-1H-benzo[d]imidazole-2-yl)quinolin-2(1H)-one(also known as dovitinib or TKI-258). AZD4547 (Gavine et al., 2012,Cancer Research 72, 2045-56,N-[5-[2-(3,5-Dimethoxyphenyl)ethyl]-2H-pyrazol-3-yl]-4-(3R,5S)-diemthylpiperazin-1-yl)benzamide),Ponatinib (AP24534; Gozgit et al., 2012, Mol Cancer Ther., 11; 690-99;3-[2-(imidazo[1,2-b]pyridazin-3-yl)ethynyl]-4-methyl-N-{4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl}benzamide,CAS 943319-70-8)

Pharmaceutical Compositions

To prepare pharmaceutical or sterile compositions includingimmunoconjugates, the immunoconjugates of the present disclosure aremixed with a pharmaceutically acceptable carrier or excipient. Thecompositions can additionally contain one or more other therapeuticagents that are suitable for treating or preventing cancergastrointestinal stromal tumors (GIST), small cell lung cancer (SCLC),acute myelod leukemia (AML), melanoma, mast cell leukemia (MCL),mastocytosis, neurofibromatosis, breast cancer, non-small cell lungcancer (NSCLC) and pancreatic cancer.

Formulations of therapeutic and diagnostic agents can be prepared bymixing with physiologically acceptable carriers, excipients, orstabilizers in the form of, e.g., lyophilized powders, slurries, aqueoussolutions, lotions, or suspensions (see, e.g., Hardman et al., Goodmanand Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, NewYork, N.Y., 2001; Gennaro, Remington: The Science and Practice ofPharmacy, Lippincott, Williams, and Wilkins, New York, N.Y., 2000; Avis,et al. (eds.), Pharmaceutical Dosage Forms: Parenteral Medications,Marcel Dekker, NY, 1993; Lieberman, et al. (eds.), Pharmaceutical DosageForms: Tablets, Marcel Dekker, NY, 1990; Lieberman, et al. (eds.)Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY, 1990;Weiner and Kotkoskie, Excipient Toxicity and Safety, Marcel Dekker,Inc., New York, N.Y., 2000).

In a specific aspect, the clinical service form (CSF) of the antibodydrug conjugates of the present disclosure is a lyophilisate in vialcontaining the ADC, sodium succinate, and polysorbate 20. Thelyophilisate can be reconstitute with water for injection, the solutioncomprises the ADC, sodium succinate, sucrose, and polysorbate 20 at a pHof about 5.0. For subsequent intravenous administration, the obtainedsolution will usually be further diluted into a carrier solution.

Selecting an administration regimen for a therapeutic depends on severalfactors, including the serum or tissue turnover rate of the entity, thelevel of symptoms, the immunogenicity of the entity, and theaccessibility of the target cells in the biological matrix. In certainaspects, an administration regimen maximizes the amount of therapeuticdelivered to the patient consistent with an acceptable level of sideeffects. Accordingly, the amount of biologic delivered depends in parton the particular entity and the severity of the condition beingtreated. Guidance in selecting appropriate doses of antibodies,cytokines, and small molecules are available (see, e.g., Wawrzynczak,Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK, 1996;Kresina (ed.), Monoclonal Antibodies, Cytokines and Arthritis, MarcelDekker, New York, N.Y., 1991; Bach (ed.), Monoclonal Antibodies andPeptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, N.Y.,1993; Baert et al., New Engl. J. Med. 348:601-608, 2003; Milgrom et al.,New Engl. J. Med. 341:1966-1973, 1999; Slamon et al., New Engl. J. Med.344:783-792, 2001; Beniaminovitz et al., New Engl. J. Med. 342:613-619,2000; Ghosh et al., New Engl. J. Med. 348:24-32, 2003; Lipsky et al.,New Engl. J. Med. 343:1594-1602, 2000).

Determination of the appropriate dose is made by the clinician, e.g.,using parameters or factors known or suspected in the art to affecttreatment or predicted to affect treatment. Generally, the dose beginswith an amount somewhat less than the optimum dose and it is increasedby small increments thereafter until the desired or optimum effect isachieved relative to any negative side effects. Important diagnosticmeasures include those of symptoms of, e.g., the inflammation or levelof inflammatory cytokines produced.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions antibody drug conjugates can be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentdisclosure employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors known in the medical arts.

Compositions comprising antibodies or fragments thereof can be providedby continuous infusion, or by doses at intervals of, e.g., one day, oneweek, or 1-7 times per week. Doses can be provided intravenously,subcutaneously, topically, orally, nasally, rectally, intramuscular,intracerebrally, or by inhalation. A specific dose protocol is oneinvolving the maximal dose or dose frequency that avoids significantundesirable side effects.

For the immunoconjugates of the present disclosure, the dosageadministered to a patient may be 0.0001 mg/kg to 100 mg/kg of thepatient's body weight. The dosage may be between 0.0001 mg/kg and 20mg/kg, 0.0001 mg/kg and 10 mg/kg, 0.0001 mg/kg and 5 mg/kg, 0.0001 and 2mg/kg, 0.0001 and 1 mg/kg, 0.0001 mg/kg and 0.75 mg/kg, 0.0001 mg/kg and0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg, 0.0001 to 0.15 mg/kg, 0.0001 to0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kg or 0.01 to 0.10 mg/kgof the patient's body weight. The dosage of the antibodies or fragmentsthereof can be calculated using the patient's weight in kilograms (kg)multiplied by the dose to be administered in mg/kg.

Doses of the immunoconjugates the can be repeated and theadministrations may be separated by at least 1 day, 2 days, 3 days, 5days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months,or at least 6 months. In a specific aspect, doses of theimmunoconjugates of the present disclosure are repeated every 3 weeks.

An effective amount for a particular patient may vary depending onfactors such as the condition being treated, the overall health of thepatient, the method, route and dose of administration and the severityof side effects (see, e.g., Maynard et al., A Handbook of SOPs for GoodClinical Practice, Interpharm Press, Boca Raton, Fla., 1996; Dent, GoodLaboratory and Good Clinical Practice, Urch Publ., London, UK, 2001).

The route of administration may be by, e.g., topical or cutaneousapplication, injection or infusion by intravenous, intraperitoneal,intracerebral, intramuscular, intraocular, intraarterial,intracerebrospinal, intralesional, or by sustained release systems or animplant (see, e.g., Sidman et al., Biopolymers 22:547-556, 1983; Langeret al., J. Biomed. Mater. Res. 15:167-277, 1981; Langer, Chem. Tech.12:98-105, 1982; Epstein et al., Proc. Natl. Acad. Sci. USA82:3688-3692, 1985; Hwang et al., Proc. Natl. Acad. Sci. USA77:4030-4034, 1980; U.S. Pat. Nos. 6,350,466 and 6,316,024). Wherenecessary, the composition may also include a solubilizing agent or alocal anesthetic such as lidocaine to ease pain at the site of theinjection, or both. In addition, pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968, 5,985,320,5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078;and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO98/31346, and WO 99/66903, each of which is incorporated herein byreference their entirety.

A composition of the present disclosure can also be administered via oneor more routes of administration using one or more of a variety ofmethods known in the art. As will be appreciated by the skilled artisan,the route and/or mode of administration will vary depending upon thedesired results. Selected routes of administration for theimmunoconjugates include intravenous, intramuscular, intradermal,intraperitoneal, subcutaneous, spinal or other parenteral routes ofadministration, for example by injection or infusion. Parenteraladministration may represent modes of administration other than enteraland topical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion. Alternatively, a composition of the present disclosure can beadministered via a non-parenteral route, such as a topical, epidermal ormucosal route of administration, for example, intranasally, orally,vaginally, rectally, sublingually or topically. In one aspect, theimmunoconjugates of the present disclosure are administered by infusion.In another aspect, the immunoconjugates are administered subcutaneously.

If the immunoconjugates of the present disclosure are administered in acontrolled release or sustained release system, a pump may be used toachieve controlled or sustained release (see Langer, supra; Sefton, CRCCrit. Ref Biomed. Eng. 14:20, 1987; Buchwald et al., Surgery 88:507,1980; Saudek et al., N. Engl. J. Med. 321:574, 1989). Polymericmaterials can be used to achieve controlled or sustained release of thetherapies of the immunoconjugates (see e.g., Medical Applications ofControlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla.,1974; Controlled Drug Bioavailability, Drug Product Design andPerformance, Smolen and Ball (eds.), Wiley, New York, 1984; Ranger andPeppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61, 1983; see alsoLevy et al., Science 228:190, 1985; During et al., Ann. Neurol. 25:351,1989; Howard et al., J. Neurosurg. 7 1:105, 1989; U.S. Pat. No.5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat.No. 5,989,463; U.S. Pat. No. 5,128,326; PCT Publication No. WO 99/15154;and PCT Publication No. WO 99/20253. Examples of polymers used insustained release formulations include, but are not limited to,poly(-hydroxy ethyl methacrylate), poly(methyl methacrylate),poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylicacid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone),poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides(PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In oneaspect, the polymer used in a sustained release formulation is inert,free of leachable impurities, stable on storage, sterile, andbiodegradable. A controlled or sustained release system can be placed inproximity of the prophylactic or therapeutic target, thus requiring onlya fraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138, 1984).

Controlled release systems are discussed in the review by Langer,Science 249:1527-1533, 1990). Any technique known to one of skill in theart can be used to produce sustained release formulations comprising oneor more immunoconjugates of the present disclosure. See, e.g., U.S. Pat.No. 4,526,938, PCT publication WO 91/05548, PCT publication WO 96/20698,Ning et al., Radiotherapy & Oncology 39:179-189, 1996; Song et al., PDAJournal of Pharmaceutical Science & Technology 50:372-397, 1995; Cleeket al., Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854, 1997;and Lam et al., Proc. Int'l. Symp. Control Rel. Bioact. Mater.24:759-760, 1997, each of which is incorporated herein by reference intheir entirety.

If the immunoconjugates of the disclosure are administered topically,they can be formulated in the form of an ointment, cream, transdermalpatch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, orother form well-known to one of skill in the art. See, e.g., Remington'sPharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms,19th ed., Mack Pub. Co., Easton, Pa. (1995). For non-sprayable topicaldosage forms, viscous to semi-solid or solid forms comprising a carrieror one or more excipients compatible with topical application and havinga dynamic viscosity, in some instances, greater than water are typicallyemployed. Suitable formulations include, without limitation, solutions,suspensions, emulsions, creams, ointments, powders, liniments, salves,and the like, which are, if desired, sterilized or mixed with auxiliaryagents (e.g., preservatives, stabilizers, wetting agents, buffers, orsalts) for influencing various properties, such as, for example, osmoticpressure. Other suitable topical dosage forms include sprayable aerosolpreparations wherein the active ingredient, in some instances, incombination with a solid or liquid inert carrier, is packaged in amixture with a pressurized volatile (e.g., a gaseous propellant, such asfreon) or in a squeeze bottle. Moisturizers or humectants can also beadded to pharmaceutical compositions and dosage forms if desired.Examples of such additional ingredients are well-known in the art.

If the compositions comprising the immunoconjugates are administeredintranasally, it can be formulated in an aerosol form, spray, mist or inthe form of drops. In particular, prophylactic or therapeutic agents foruse according to the present disclosure can be conveniently delivered inthe form of an aerosol spray presentation from pressurized packs or anebuliser, with the use of a suitable propellant (e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridges(composed of, e.g., gelatin) for use in an inhaler or insufflator may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

Methods for co-administration or treatment with a second therapeuticagent, e.g., a cytokine, steroid, chemotherapeutic agent, antibiotic, orradiation, are known in the art (see, e.g., Hardman et al., (eds.)(2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics,10.sup.th ed., McGraw-Hill, New York, N.Y.; Poole and Peterson (eds.)(2001) Pharmacotherapeutics for Advanced Practice:A Practical Approach,Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo (eds.)(2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams &Wilkins, Phila., Pa.). An effective amount of therapeutic may decreasethe symptoms by at least 10%; by at least 20%; at least about 30%; atleast 40%, or at least 50%.

Additional therapies (e.g., prophylactic or therapeutic agents), whichcan be administered in combination with the immunoconjugates of theinvention may be administered less than 5 minutes apart, less than 30minutes apart, 1 hour apart, at about 1 hour apart, at about 1 to about2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hoursto about 4 hours apart, at about 4 hours to about 5 hours apart, atabout 5 hours to about 6 hours apart, at about 6 hours to about 7 hoursapart, at about 7 hours to about 8 hours apart, at about 8 hours toabout 9 hours apart, at about 9 hours to about 10 hours apart, at about10 hours to about 11 hours apart, at about 11 hours to about 12 hoursapart, at about 12 hours to 18 hours apart, 18 hours to 24 hours apart,24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120hours apart from the immunoconjugates of the present disclosure. The twoor more therapies may be administered within one same patient visit.

In certain aspects, immunoconjugates can be formulated to ensure properdistribution in vivo. For example, the blood-brain barrier (BBB)excludes many highly hydrophilic compounds. To ensure that thetherapeutic compounds of the disclosure cross the BBB (if desired), theycan be formulated, for example, in liposomes. For methods ofmanufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548;and 5,399,331. The liposomes may comprise one or more moieties which areselectively transported into specific cells or organs, thus enhancetargeted drug delivery (see, e.g., Ranade, (1989) J. Clin. Pharmacol.29:685). Exemplary targeting moieties include folate or biotin (see,e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides (Umezawa etal., (1988) Biochem. Biophys. Res. Commun 153:1038); antibodies (Bloemanet al., (1995) FEBS Lett. 357:140; Owais et al., (1995) Antimicrob.Agents Chemother. 39:180); surfactant protein A receptor (Briscoe etal., (1995) Am. J. Physiol. 1233:134); p 120 (Schreier et al, (1994) J.Biol. Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBSLett. 346:123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273.

The present disclosure provides protocols for the administration ofpharmaceutical composition comprising immunoconjugates alone or incombination with other therapies to a subject in need thereof. Thetherapies (e.g., prophylactic or therapeutic agents) of the combinationtherapies of the present invention can be administered concomitantly orsequentially to a subject. The therapy (e.g., prophylactic ortherapeutic agents) of the combination therapies of the presentinvention can also be cyclically administered. Cycling therapy involvesthe administration of a first therapy (e.g., a first prophylactic ortherapeutic agent) for a period of time, followed by the administrationof a second therapy (e.g., a second prophylactic or therapeutic agent)for a period of time and repeating this sequential administration, i.e.,the cycle, in order to reduce the development of resistance to one ofthe therapies (e.g., agents) to avoid or reduce the side effects of oneof the therapies (e.g., agents), and/or to improve, the efficacy of thetherapies.

The therapies (e.g., prophylactic or therapeutic agents) of thecombination therapies of the disclosure can be administered to a subjectconcurrently.

The term “concurrently” is not limited to the administration oftherapies (e.g., prophylactic or therapeutic agents) at exactly the sametime, but rather it is meant that a pharmaceutical compositioncomprising antibodies or fragments thereof are administered to a subjectin a sequence and within a time interval such that the immunoconjugatescan act together with the other therapy(ies) to provide an increasedbenefit than if they were administered otherwise. For example, eachtherapy may be administered to a subject at the same time orsequentially in any order at different points in time; however, if notadministered at the same time, they should be administered sufficientlyclose in time so as to provide the desired therapeutic or prophylacticeffect. Each therapy can be administered to a subject separately, in anyappropriate form and by any suitable route. In various aspects, thetherapies (e.g., prophylactic or therapeutic agents) are administered toa subject less than 15 minutes, less than 30 minutes, less than 1 hourapart, at about 1 hour apart, at about 1 hour to about 2 hours apart, atabout 2 hours to about 3 hours apart, at about 3 hours to about 4 hoursapart, at about 4 hours to about 5 hours apart, at about 5 hours toabout 6 hours apart, at about 6 hours to about 7 hours apart, at about 7hours to about 8 hours apart, at about 8 hours to about 9 hours apart,at about 9 hours to about 10 hours apart, at about 10 hours to about 11hours apart, at about 11 hours to about 12 hours apart, 24 hours apart,48 hours apart, 72 hours apart, or 1 week apart. In other aspects, twoor more therapies (e.g., prophylactic or therapeutic agents) areadministered to a within the same patient visit.

The prophylactic or therapeutic agents of the combination therapies canbe administered to a subject in the same pharmaceutical composition.Alternatively, the prophylactic or therapeutic agents of the combinationtherapies can be administered concurrently to a subject in separatepharmaceutical compositions. The prophylactic or therapeutic agents maybe administered to a subject by the same or different routes ofadministration.

EXAMPLES Example 1 Generation of cKIT Abs by Hybridoma TechnologyAntigen and Other Proteins

A transient expression cell line secreting human cKIT protein wasgenerated by transfection of 293 Freestyle™ cells (Invitrogen, Carlsbad,Calif.). Briefly, the cells cultivated in Freestyle™ medium (Invitrogen)were transfected using 293Fectin™ transfection reagent and a recombinantplasmid containing the ECD of the human cKIT cDNA and either a His6 tagat the C-terminus of the sequence, or a murine Fc (pFUSE, Invivogen, SanDiego, Calif.). 48-72 hours later the media is centrifuged to remove thecells, sterile filtered, and the cleared lysate used for proteinpurification.

For the His6 tagged cKIT: The resulting concentrate was applied to aNiNTA His-Bind Superflow column at 0.5 mL/min. After baseline washingwith PBS, bound material was eluted with PBS with a stepwise gradient ofImidazole (10-500 mM). The resulting eluate was dialyzed against PBS, pH7.3, sterile filtered and aliquotted. For Fc-cKit fusion, Protein GfastFlow columns were used (instead of NiNTA) as outlined above, andeluted with a pH 3 Glycine buffer, which was neutralized with Tris, pH8.

Hybridoma Generation Immunization of Mice and Production of Hybridomas

Purified cKIT was diluted 1:1 with Freunds Complete Adjuvant prior toimmunization of Bcl-2 transgenic mice (C57BL/6-Tgn (bcl-2) 22 Wehistrain). Mice were immunized using a procedure that calls for RepetitiveImmunization at Multiple Sites (RIMMS) (McIntyre G D., Hybridoma, 1997).Briefly, mice were injected with 1-3 μg of antigen at 8 specific sitesproximal to peripheral lymph nodes (PLN). This procedure was repeated 8times over a 12-day period. On Day 12, a test bleed was collected andthe serum antibody titer was analyzed by ELISA. Pooled PLN were removedfrom high titer mice on Day 15. To harvest lymphocytes, PLN were washedtwice with plain DMEM and then dissociated by passage through a 0.22micron screen (Falcon #352350, BD Bioscience, San Jose, Calif.). Theresulting lymphocytes were washed 2 additional times prior to fusion. F0myeloma cells were mixed with lymphocytes at a ratio of 2.5 lymphocytesto 1 F0 cell. The cell mixture was centrifuged and 1 mL of PEG 1500 wassubsequently added dropwise to the cell pellet for 1 min. After 30seconds, 1 mL of DMEM was slowly added, and 1 min later, 19 mL of DMEMwas added for 5 min. Fused cells were pelleted, suspended at a densityof 2×10⁵ cells/mL in HAT media (DMEM+20% FBS, Pen/Strep/Glu, 1×NEAA,1×HAT, 0.5×HFCS), and placed at 37° C. for one hr. The cells were thenplated in 384-well plates at 60 μL/well.

Screening of Hybridomas Secreting Antibodies to cKIT

Ten days after fusion, hybridoma plates were screened for the presenceof cKIT specific antibodies. For the ELISA screen, Maxisorp 384-wellplates (Nunc #464718) were coated with 50 μL of cKIT (diluted to 15ng/well in PBS) and incubated overnight at 4° C. The remaining proteinwas aspirated and wells were blocked with 1% BSA in PBS. After 30 minincubation at room temperature, the wells were washed four times withPBS+0.05% Tween (PBST). 15 μL of hybridoma supernatant was transferredto the ELISA plates. 15 μL of mouse serum, taken at the time of PLNremoval, was diluted 1:1000 in PBS and added as a positive control. 50μL of secondary antibody (goat anti mouse IgG-HRP (Jackson ImmunoResearch #115-035-071, West Grove, Pa.), diluted 1:5000 in PBS) wasadded to all wells on the ELISA plates. After incubation at roomtemperature for 1 h, the plates were washed eight times with PBST. 25 μLof TMB (KPL #50-76-05) was added and after 30 min incubation at roomtemperature; the plates were read at an absorbance of 605 nm. Cells frompositive wells were expanded into 24-well plates in HT media (DMEM+20%FBS, Pen/Strep/Glu, 1×NEAA, 1×HT, 0.5×HFCS).

Antibody Purification

Supernatant containing cKIT antibodies were purified using protein G(Upstate #16-266 (Billerica, Mass.)). Prior to loading the supernatant,the resin was equilibrated with 10 column volumes of PBS. Followingbinding of the sample, the column was washed with 10 column volumes ofPBS, and the antibody was then eluted with 5 column volumes of 0.1 MGlycine, pH 2.0. Column fractions were immediately neutralized with1/10th volume of Tris HCl, pH 9.0. The OD280 of the fractions wasmeasured, and positive fractions were pooled and dialyzed overnightagainst PBS, pH 7.2.

Example 2 Humanization and Affinity Maturation of Anti-cKIT AntibodiesDesign of Humanization

VH and VL sequences of hybridoma derived anti-cKIT antibody 9P3 are SEQID NO:9 and SEQ ID NO.18, respectively. Amino acid sequences of humanIgG1 constant domains used to generate full IgG1 are SEQ ID NO.10 forthe heavy chain and SEQ ID NO.19 for the light chain. Humanization ofthe heavy chain was accomplished by grafting the 3 CDR regions(GFTFSDYYMA (SEQ ID NO. 148,)) (NINYDGSSTYYLDS (SEQ ID NO.149)) and(GDYYGTTYWYFDV (SEQ ID NO:150) from anti-cKIT antibody 9P3, onto humangermline acceptor framework VH3_(—)3-07 (vBASE database). Humanizationof the light chain was accomplished by either grafting the 3 CDR regions(RASQDISNYLN (SEQ ID NO.151)), (YTSRLQS (SEQ ID NO.152)) and (QQGKKLWS(SEQ ID NO:153)) from anti-cKIT antibody 9P3, onto human germlineacceptor framework VK3-L25 (vBASE database) or grafting 2 CDR regions(SEQ ID NO.152 and SEQ ID NO.153) onto human germline acceptor frameworkVK1-012 (vBASE database). In addition to the CDR regions, one frameworkresidue of the variable light chain domain, i.e. VL #71 and in the caseof VK3-L25 VL #79 (residue numbering based on SEQ ID NO.21) was retainedfrom the 9P3 sequence. Further, the human J elements JH4 and JK4 wereused for the heavy and light chain, respectively. The resulting aminoacid sequences of the humanized antibody heavy chain is SEQ ID NO. 11and for the two light chains SEQ ID NO. 20 (VK1-012) and SEQ ID NO. 21(VK3-L6).

We hypothesized that the amino acid motif aspartate followed by glycine(DG) may be susceptible to post-translational modification(iso-aspartate formation) and that lysines within the CDRs may decreasethe fraction of active antibody after antibody-drug conjugation.Further, we intended to affinity mature the humanized antibody. Acombination of random mutagenesis (i.e. error-prone PCR) and directedmutagenesis was applied to optimize the humanized antibodies.

Generation of Humanized Sequences

DNA sequences coding for humanized VL and VH domains were ordered atGeneArt (Life Technologies Inc. Regensburg, Germany) including codonoptimization for homo sapiens. Sequences coding for VL and VH domainswere subcloned by cut and paste from the GeneArt derived vectors intoexpression vectors suitable for secretion in mammalian cells. The heavyand light chains were cloned into individual expression vectors to allowco-transfection. Elements of the expression vector include a promoter(Cytomegalovirus (CMV) enhancer-promoter), a signal sequence tofacilitate secretion, a polyadenylation signal and transcriptionterminator (Bovine Growth Hormone (BGH) gene), an element allowingepisomal replication and replication in prokaryotes (e.g. SV40 originand ColE1 or others known in the art) and elements to allow selection(ampicillin resistance gene and zeocin marker).

Expression and Purification of Humanized Antibodies

Human Embryonic Kidney cells constitutively expressing the SV40 large Tantigen (HEK293-T ATCC11268) are one of the preferred host cell linesfor transient expression of humanized and/or optimized IgG proteins.Transfection is performed using PEI (Polyethylenimine, MW 25.000 linear,Polysciences, USA Cat. No. 23966) as transfection reagent. The PEI stocksolution is prepared by carefully dissolving 1 g of PEI in 900 ml cellculture grade water at room temperature (RT). To facilitate dissolutionof PEI, the solution is acidified by addition of HCl to pH 3-5, followedby neutralization with NaOH to a final pH of 7.05. Finally, the volumeis adjusted to 1 L and the solution is filtered through a 0.22 μmfilter, aliquotted and frozen at −80° C. until further use. Once thawed,an aliquot can be re-frozen up to 3 times at −20° C. but should not bestored long term at −20° C. HEK 293T cells are cultivated using aNovartis proprietary serum-free culture medium for transfection andpropagation of the cells, and ExCell VPRO serum-free culture medium(SAFC Biosciences, USA, Cat. No. 24561C) as production/feed medium.Cells prepared for transient transfections are cultivated in suspensionculture. For small scale (<5 L) transfections, cells are grown inCorning shake flasks (Corning, Tewksbury, Mass.) on an orbital shaker(100-120 rpm) in a humidified incubator at 5% CO2 (seed flasks). Cellsin the seed cultures should be maintained in the exponential growthphase (cell densities between 5×10⁵ and 3×10⁶/mL) and display aviability of >90% for transfection. Cell densities outside of this rangewill result in either a lag phase after dilution or reduced transfectionefficiency. For small scale (<5 L) transfection an aliquot of cells istaken out of the seed cultures and adjusted to 1.4×10⁶ cells/mL in 36%of the final volume with Novartis serum-free culture medium. The DNAsolution (Solution 1: 0.5 mg of heavy chain and 0.5 mg of light chainexpression plasmid for a 1 L transfection) is prepared by diluting theDNA to 1 mg/L (final volume) in 7% of the final culture volume followedby gentle mixing. To prevent bacterial contamination, this solution isfiltered using a 0.22 μm filter (e.g. Millipore Stericup). Then 3 mg/L(final volume) of PEI solution is also diluted in 7% of final culturevolume and mixed gently (Solution 2). Both solutions are incubated for5-10 min at room temperature (RT). Thereafter solution 2 is added tosolution 1 with gentle mixing and incubated for another 5-15 minutes atroom temperature. The transfection mix is then added to the cells andthe cultivation of cells is continued for 4 to 6 hours. Finally, theremaining 50% of total production volume are achieved by addition ofExCell® VPRO serum-free culture medium. The cell cultivation iscontinued for eleven days post transfection. The culture is harvested bycentrifugation at 4500 rpm for 20 minutes at 4° C. (Heraeus®, Multifuge3S-R, Thermo Scientific, Rockford, Ill.). The cell supernatant recoveredis sterile filtered through a stericup filter (0.22 μm) and stored at 4°C. until further processing.

Purification was performed on an “ÄKTA 100 explorer Air” chromatographysystem at 4° C. in a cooling cabinet, using a freshly sanitized (0.25 MNaOH) HiTrap ProtA MabSelect®SuRe, 5 ml column. The column wasequilibrated with 5 CV of PBS (Gibco, Life Technologies, Carlsbad,Calif.), and then the sterile filtered supernatant (2 L) was loaded at4.0 ml/min. The column was washed with 8 CV of PBS to elute the unboundsample and again washed with 5 CV of PBS. Antibody was eluted with 5 CVof 50 mM citrate, 70 mM NaCl pH 3.2. The eluate was collected in 3 mlfractions; fractions were pooled and adjusted at pH 7 with 1 M Tris HClpH10. The pools were pooled and sterile filtered (Millipore Steriflip,0.22 um), the OD 280 nm was measured in a Spectrophotometer ND-1000(NanoDrop), and the protein concentration was calculated based on thesequence data. The eluate was tested for aggregation (SEC-MALS) andpurity (SDS-PAGE, LAL and MS). For the second purification step, ifneeded, pools from the first purification were loaded into a freshlysanitised (0.5 M NaOH)SPX (Hi Load 16/60 Superdex 200 grade 120 mL(GE-Helthcare). The column was equilibrated with PBS and the run wasdone with PBS buffer at 1 ml/min, the eluate was collected in 1.2 mlfractions and analyzed as described for the first purification step.

Example 3 Screening for Anti-cKIT Antibodies HuCAL PLATINUM™ Pannings

For selection of antibodies recognizing human cKIT multiple panningstrategies were employed. Therapeutic antibodies against human cKITproteins were generated by selection of clones having high affinitybinding affinities, using as the source of antibody variant proteins acommercially available phage display library, the Morphosys HuCALPLATINUM® library (Morphosys, Munich Del.). The phagemid library isbased on the HuCAL® concept (Knappik et al., (2000) J Mol Biol 296:57-86) and employs the CysDisplay® technology for displaying the Fab onthe phage surface (Lohning, WO 01/05950). For isolation of anti-cKITantibodies, standard panning strategies were performed using solidphase, solution, whole cell and differential whole cell panningapproaches.

Solid Phase Panning Against cKIT

An 96-well Maxisorp™ plate was coated with human or mouse cKIT Fc fusionprotein o/n at 4° C. For each panning, about 4×10¹³ HuCAL PLATINUM®phage-antibodies were added to each antigen coated and incubated for 2 hat RT on a microtiter plate shaker. Afterwards, unspecific bound phageswere washed off by several washing steps and specifically bound phages,were eluted using 25 mM DTT in 10 mM Tris/HCl pH 8.

The eluate was transferred into 14 ml of E. coli bacteria and incubatedfor phage infection. The infected bacteria were resuspended in 2×YTmedium, plated on LB/Cam agar plates and incubated o/n. Colonies werescraped off the plates and were used for phage rescue, polyclonalamplification of selected clones, and phage production. With purifiedphage the next panning round was started.

The second and third round of solid phase panning was performedaccording to the protocol of the first round except for decreasedamounts of antigen and more stringent washing conditions.

Capture Panning Against cKIT

For capture panning, the antigen cKIT/murine Fc fusion proteins wereimmobilized on a 96-well Maxisorp™ plate via an goat anti-mouse Fccapture antibody. During phage blocking, human and mouse γ globulin wereadded to the blocking buffer to avoid selection of antibodies againstthe capture antibody and mouse Fc part of the antigen. The antigencoating and phage blocking procedures in the capture panning wasperformed as described in the Solid Phase Panning protocol (see above).

Solution Panning Protocol with Streptavidin-Coupled Magnetic Beads

Solution pannings were performed in two different modes (“classical” and“alternative”). For each panning, about 4×10¹³ HuCAL PLATINUM®phage-antibodies were blocked with an equal volume of 2×Chemiblocker/0.1% Tween20. For removal of Streptavidin- or bead-bindingphage, pre-adsorption of blocked phage particles was performed twiceusing 1 mg blocked Streptavidin beads each.

a) “Classical” mode: Biotinylated 16P23 mAb was incubated with humancKIT ECD-H is protein and was added to the blocked phage particles. The16P23 antibody is an internally generated hybridoma and was used invarious screening protocols as a capture antibody to expose differentdomains on the ECD of cKIT. The 16P23 antibody was also used forantibody binning purposes. After incubation the phage-antigen complexeswere captured using Streptavidin beads and phage particles bound to theStreptavidin beads were collected with a magnetic separator.

b) “Alternative” mode: Biotinylated 16P23 mAb was added to streptavidinbeads and the antibody-bead mix was incubated on a rotator at RT for 30min. Beads were washed and resuspended in PBS containing human cKITECD-His protein. Subsequently, the phages were added and theantibody-bead-antigen-phage complex was rotated for an additional 1 h atRT on a rotator. After this last incubation step the beads were capturedwith a magnetic separator and the supernatants were discarded.

Using both display methods unspecific bound phage were washed off byseveral washing steps using PBS/0.05% Tween20 and PBS. Specificallybound phages were eluted from Streptavidin beads by using 25 mM DTT in10 mM Tris/HCl pH 8. Subsequent phage infection and phage production wasperformed according to the Solid Phase Panning protocol.

The second and third round of the solution panning was performedaccording to the protocol of the first round except for decreasedamounts of antigen and more stringent washing conditions.

Whole Cell Panning Against cKIT

Target cells expressing antigen human, mouse or rat cKIT were used asantigens and were contacted with HuCAL PLATINUM® phage-antibodies forpannings. The phage-cell complexes were washed three times in PBS/5%FCS. Elution of specifically bound phage from target cells was performedwith 0.1 M glycine-HCl/0.5 M NaCl, pH 2.2. Subsequent phage infectionand phage production was performed according to the Solid Phase Panningprotocol. The second and third round of the whole cell panning wasperformed according to the protocol of the first round.

Differential Whole Cell Panning Against cKIT

In the differential whole cell panning, the selection was donealternating on cells and purified protein. The selection rounds onpurified antigen were performed as described in the Solid Phase Panningprotocol For the selection rounds on cells please refer to the procedurein the Whole Cell Panning Against cKIT section.

Maturation Pannings

In order to obtain specific antibodies with increased affinities,maturation pannings were performed (Prassler et al., Future Med. Immuno.2009 1(4):571-583). For this purpose, sequenced clones already testedfor cKIT specific binding were used for LCDR3 or HCDR2 cassetteexchange. Afterwards two rounds of solid phase pannings were performedwith human and/or mouse cKIT Fc fusion protein as described in the SolidPhase Panning protocol.

a) For LCDR3RapMAT®: Fab-encoding fragments of phage derived pMORPH30®vector DNA (Morphosys, Munich Del.) were enzymatically digested andinserts were replaced with TRIM™ LCDR3 maturation cassettes (Virnekaeset al., NAR 1994 22(25):5600-5607). Subsequently, 1.25 μg pMORPH30®display vector was ligated with the insert fragment carrying thediversified LCDR3 s.

b) For HCDR2RapMAT®: After the 2nd round of panning, Fab-encodingfragments of phage derived pMORPH30® vector DNA were enzymaticallydigested and inserts were replaced with TRIM™ HCDR2 maturation cassettes(Virnekas et al., supra). Subsequently, 1.25 μg pMORPH30® display vectorwas ligated with the insert fragment carrying the diversified HCDR2s.

The generated libraries were amplified and subjected to two rounds ofpanning with either increased stringency and reduced antigenconcentration or alternation of human and mouse cKIT antigen to identifyaffinity improved clones.

Preparation of Fab Containing Bacterial Lysates for ELISA Screening

For initial screening and characterization an o/n culture of individualFab-expressing E. coli clones were lysed using lysozyme, 4 mM EDTA and10 U/μl Benzonase. Fab containing E. coli lysates were used for ELISA,FACS and SET screening.

Screening of Fab-Containing Raw Bacterial Lysates ELISA Screening

Using ELISA screening, single Fab clones are identified from panningoutput for binding to the target antigen. Fabs are tested using Fabcontaining crude E. coli lysates.

Fab Expression Check ELISA

For verification of Fab expression in the prepared E. coli lysates,Maxisorp™ 384 well plates (Nunc, Sigma-Aldrich, St.Louis Mo.) werecoated with Fd fragment specific sheep anti-human IgG diluted 1:1000 inPBS. After blocking with 5% skim milk powder in PBS containing 0.05%Tween20, Fab-containing E. coli lysates were added. Subsequently thebound HuCAL®-Fab fragments were detected by incubation with F(ab)₂specific goat anti-human IgG conjugated to alkaline phosphatase (diluted1:5000) followed by addition of AttoPhos® fluorescence substrate (Roche,#11681982001, Mannheim, Del.). Fluorescence emission at 535 nm wasrecorded with excitation at 430 nm.

ELISA Screening on Directly Coated Antigen

Maxisorp™ 384 well plates were coated with mFc tagged human cKIT ECDprotein at a concentration of 10 μg/ml in PBS. After blocking of plateswith 5% skim milk powder in PBS, Fab-containing E. coli lysates wereadded. Binding of Fabs was detected by F(ab)₂ specific goat anti-humanIgG conjugated to alkaline phosphatase (diluted 1:5000) using Attophos®fluorescence substrate (Roche, #11681982001, Mannheim, Del.).Fluorescence emission at 535 nm was recorded with excitation at 430 nm.

Epitope Binning with Fab BEL Lysates

To identify potential ligand binding competitors prior to affinitymaturation, a competition ELISA screening with Fab E. coli lysates and16P23 antibody, a known ligand binding competitor, was performed. Forthis purpose, Maxisorp™ 384 well plates were coated with mFc taggedhuman cKIT ECD protein and blocked as described above (ELISA Screeningon Directly Coated Antigen).

16P23 mAb was added at a final concentration of 5 μg/ml followed byincubation with Fab-containing E. coli lysates. Finally, binding of Fabswas detected with an anti-FLAG alkaline phosphatase-conjugated antibody(Sigma A-9469, diluted to 1:10000) using Attophos® fluorescencesubstrate (Roche, #11681982001). Fluorescence emission at 535 nm wasrecorded with excitation at 430 nm.

FACS Screening

In FACS screening, single Fab clones binding to cell surface expressedantigen are identified from the panning output. Fabs are tested usingFab containing crude E. coli lysates.

FACS Screening was performed either in 96- or 384-well plate format:

a) In 96-well plate format using a BD FACS array device, 100 μl ofcell-suspension were transferred into a fresh 96-well plate (resultingin 1×10⁵ cells/well). Target cell suspension containing plate wascentrifuged and supernatant was discarded. Remaining cell pellet wasresuspended and 50 μl of Fab containing BEL extracts was added to thecorresponding wells. Plate was incubated on ice for 1 hour. Followingincubation, cells were spun down and washed three times with 200 μl FACSbuffer (PBS, 3% FCS). After each washing step, cells were centrifugedand carefully resuspended. Secondary detection antibody (PE conjugatedgoat anti human IgG; Dianova, Hamburg, Del.) was added and samples wereincubate on ice and subsequently washed according to Fab incubation.Finally, cell pellets were resuspended in 150 μl FACS buffer per welland samples were analyzed in BD FACS array.

b) In 384-well plate format using a BD Calibur® HTS device (BDBiosciences, San Jose, Calif.), 20 μl of cell-suspension weretransferred into a fresh 384 round well plate (resulting in 4×10⁴cells/well). Target cell suspension containing plate was centrifuged andsupernatant was discarded. Remaining cell pellet was resuspended and 20μl of Fab containing extracts was added to the corresponding wells.Plate was incubated for 1 hour shaking at 4° C. Following incubation,cells were spun down and washed three times with 40 μl FACS buffer (PBS,3% FCS). After each washing step, cells were centrifuged and carefullyresuspended. 40 μl of PE conjugated goat anti human detection antibodywas added and samples were incubated on ice and subsequently washedaccording to Fab incubation. Finally, cell pellets were resuspended in35 μl FACS buffer per well and samples were measured with BD FACSCalibur/HTS device.

Affinity Determination

For K_(D) determinations, monomer fractions of antibody protein wereused (at least 90% monomer content, analyzed by analytical SEC; Superdex75 PC3.2/30 (GE Healthcare, Pittsburgh, Pa.) for Fab, or Tosoh TSKgelG3000 SW_(XL) (7.8 mm/30.0 cm) (Tosoh Bioscience GmbH, Stuttgart, Del.)for IgG, respectively).

Solution Equilibrium Titration (SET) Method for KD Determination UsingSector Imager 6000 (MSD)

Affinity determination in solution was basically performed as describedin the literature (Friquet et al., J. Immuno. Meth. 1985; 77:305-319).In order to improve the sensitivity and accuracy of the SET method, itwas transferred from classical ELISA to ECL based technology (Haenel etal., 2005). 1 mg/ml goat-anti-human (Fab)₂ fragment specific antibodies(Dianova) were labeled with MSD Sulfo-TAG™ NHS-Ester (Meso ScaleDiscovery, Gaithersburg, Md., USA) according to the manufacturer'sinstructions. MSD plates were coated with antigen and the equilibratedsamples were transferred to those plates. After washing, 30 μl per wellof the MSD-Sulfo-tag labeled detection antibody (anti-human (Fab)₂) wasadded to the MSD plate and incubated on a shaker. After washing the MSDplate and adding 30 μl/well MSD Read Buffer T with surfactant,electrochemiluminescence signals were detected using a Sector Imager6000 (Meso Scale Discovery, Gaithersburg, Md., USA).

The data was evaluated with XLfit (IDBS) software applying customizedfitting models. For K_(D) determination of Fab molecules the followingfit model was used (according to (Haenel et al., Anal. Biochem 2005;339(1):182-184), modified according to (Abraham et al., J. Mol. Recogn1996; 9:456-461)):

$y = {B_{\max} - \left( {\frac{B_{\max}}{{2\lbrack{Fab}\rbrack}_{t}}\left( {\lbrack{Fab}\rbrack_{t} + x + K_{D} - \sqrt{\left( {\lbrack{Fab}\rbrack_{t} + x + K_{D}} \right)^{2} - {4{x\lbrack{Fab}\rbrack}_{t}}}} \right)} \right)}$

[Fab]_(t): applied total Fab concentrationx: applied total soluble antigen concentration (binding sites)B_(max): maximal signal of Fab without antigenK_(D): affinityFor K_(D) determination of IgG molecules the following fit model for IgGwas used (modified according to (Piehler et al., 1997)):

$y = {\frac{2\; B_{\max}}{\lbrack{IgG}\rbrack}\left( {\frac{\lbrack{IgG}\rbrack}{2} - \frac{\left( {\frac{x + \lbrack{IgG}\rbrack + K_{D}}{2} - \sqrt{\frac{\left( {x + \lbrack{IgG}\rbrack + K_{D}} \right)^{2}}{4} - {x\lbrack{IgG}\rbrack}}} \right)^{2}}{2\lbrack{IgG}\rbrack}} \right)}$

[IgG]: applied total IgG concentrationx: applied total soluble antigen concentration (binding sites)B_(max): maximal signal of IgG without antigenK_(D): affinity

Experimental Settings:

K_(D) determination of HuCAL® anti cKIT IgGs was basically performed asfollows: human cKIT-Fc was coated at 0.1 μg/ml in PBS o/n at 4° C. onstandard MSD plates/assay buffer for 1 h at RT on streptavidin MSDplates. Subsequently MSD plates were blocked with PBS with 3% BSA for 1h at RT. Streptavidin plates were blocked o/n at 4° C. with PBS with 5%BSA before antigen coating. For titration of antigen human cKIT-His wasapplied.

Subsequently, the concentration of unbound Fab was quantified via ECLdetection using the Sector Imager 6000 (Meso Scale Discovery,Gaithersburg, Md., USA). Results were processed using XLfit (IDBS)software, applying the corresponding fit model to estimate affinitiesand thus identify clones most improved by the maturation.

In Vitro Biochemical Assays (Cross-Reactivity and Domain-BindingAnalysis)

Purified IgGs were tested in ELISA for binding to human, cyno and mousecKIT full-length ECD proteins as well as human cKIT ECD domainconstructs D1-3 and D4-5. For this purpose plates were coated withantigen at a concentration of 5 μg/ml in PBS over night at 4° C. Bindingof IgGs was detected by anti-human or anti-mouse F(ab)₂ conjugated toalkaline phosphatase (diluted 1:5000 in 1% MPBS) using Attophos® assubstrate. Fluorescence emission was measured at an excitation of 430 nmand an emission of 535 nm.

Epitope Binning of Purified IgGs

Purified IgG candidates were tested for competition with internallygenerated tool antibodies, previously shown to define individual bins onthe extracellular domain of cKIT. For this purpose, IgGs were coated atconstant amounts on Maxisorp™ plates and tested for competition withincreasing amounts of competitor IgG in solution. As positive control,the coated IgG was analyzed for competition with itself in solution. Alltested IgGs were preincubated in 50× excess with glycobiotinylated humancKIT-Fc fusion for 1 h at RT in solution. Antigen/antibody complexeswere then added to the coated antibodies and detection of boundcomplexes occurred via the biotinylated antigen. In general, signals athigh IgG concentration could only be obtained when the coated IgG wasable to bind to accessible epitopes on the antigen different to thetested IgG in solution (i.e. a non competitive antibody). In contrast,for competitive antibodies, antibodies with partially overlappingepitopes or antibodies that block the epitope by steric hindrance,binding signals at high IgG concentration were significantly decreasedin contrast to controls.

Respective wells of Maxisorp™ plates were coated with 20 μl/well of IgGdilution at a concentration of 1.2 μg/ml in PBS, incubated overnight at4° C. and then washed 3× with PBST. Plates were blocked with 90 μl 3%BSA/PBS well for 1 h at RT and washed 3× with PBST.

EC50 Determination on Cells Via FACS

Purified IgGs were tested at a single concentration or titrated in FACSto determine EC50 values for binding to cell surface expressed human,mouse or rat cKIT. For this purpose, Mole, P815 or RBL-2H3 cells wereharvested with Accutase® (Life Technologies, Carlsbad, Calif.) anddiluted to 1×10⁶/ml in FACS buffer. All subsequent steps were done onice to prevent internalization of the receptor. The cell suspension wasfilled with 100 μl/well into a 96 well U bottom plate. Aftercentrifugation at 210 g for 5 min at 4° C., buffer was discarded. 100 μlof the specific mAbs diluted in FACS buffer was then added per well at aconcentration of 15 μg/ml or in titration experiments at a serialdilution of antibody concentrations (1:3 dilution steps, startingconcentration of 15 μg/ml). After 1 h incubation on ice, cells werewashed three times with 150 μl FACS buffer. Secondary PE conjugated goatanti human detection antibody (diluted 1:200 in FACS buffer) was addedto the cells with 100 μl/well and incubated on ice for 1 h. Cells werewashed three times with 150 μl FACS buffer. Finally, cell pellets wereresuspended in 200 μl FACS buffer per well and samples were analyzed inBD FACS array.

In Vitro Bio-Assays SCF-Dependent Proliferation Assay

Proliferation assays were performed on the Mole cell line (human acutemegakaryoblastic leukemia, DSMZ no.: ACC 104) cultured in RPMI1640 withstable glutamine (PAN #P04-18500), 10% FCS and 10 ng/ml SCF (R&DCAT#255-SC; Lot#CM2810061, R&D Corp, Berkeley Calif.).

In the SCF-dependent proliferation assay purified IgGs or IgG containingcell culture supernatants were tested. In both experimental settingscells were harvested and resuspended in 50 ml starve medium (culturemedium without SCF) at a concentration of 0.5×10⁶ cells/ml and incubatedat 37° C. for 18 h. Cells were then resuspended at a concentration of1×10⁶ cells/ml in starving medium with 60 ng/ml SCF (2× concentrated,final concentration after addition of antibody is 30 ng/ml). 50 μl ofcells (5×10⁴ cells/well) and 50 μl of 2× concentrated purifiedantibodies or undiluted cell culture supernatants were added per well ofa white 96-well flat with clear bottom plates. For negative and positivecontrols, cells w/o SCF and w/o antibody or cells with SCF and w/oantibody were included. Plates were incubated for 48 h at 37° C. andfinally cell numbers were determined using CellTiter-Glo® (Promega#G7571, Promega, Madison, Wis.) according to the manufacturer'sinstructions.

Fab-ZAP ADC Piggyback Assay

To test the ability of antibodies to internalize after receptor binding,an ADC assay was performed mixing Fab-ZAP reagent (goatanti-hu-mAb-saporin-coupled; ATS Biotechnology, Cat#IT-51-250, ATS Bio,San Diego, Calif.) either with purified IgGs or with IgG containing cellculture supernatants. Cytotoxic potential was tested on the cancer cellline CMK-11-5 (acute megakaryoblastic leukemia cells, cultured inRPMI1640+10% FCS) as these cells show high expression of cKIT.

Cells in culture were counted and diluted in medium to a concentrationof 1×10⁵ cells/ml. 50 μl cell suspension (5000 cells/well) weretransferred to 96-well plates (Flat Clear Bottom White Plate TC-Treated,Corning Cat#3903, Corning, Tewksbury, Mass.). In a separate plate (96Well V bottom, Nunc, Cat#249946, Nunc Sigma-Aldrich, St. Louis, Mo.)IgGs were diluted in medium. IgG containing cell culture supernatantswere diluted 1:125 and purified IgGs to a concentration of 0.4 nMresulting in a total volume of 60 μl/well. An equal volume of FabZAPsolution at a concentration of 5 nm was added and the plate wasincubated for 60 min at 37° C. 50 μl of antibody/Fab-ZAP conjugates weretransferred to CMK-11-5 cells (total volume 100 μl). For controls, wellswith cells only (=100% viability control) and cells only incubated withFab-ZAP (to check for unspecific killing of the secondary reagent) wereprepared. Final concentration of Fab-ZAP was 1.25 nM. Plates wereincubated for 72 h at 37° C. and 5% CO₂. Cell numbers were determinedusing CellTiter-Glo® (Promega #G7571) according to the manufacturer'sinstructions. Viability was normalized to the cells only control.

Summary

In Screening for cKIT Antibodies, 2 Different Strategies were Performed:

Strategy 1:

Candidates with human/cyno x-reactivity (217 HCDR3 families) wereselected on high affinity and after IgG conversion clones were screenedfor functionality in CMK-11-5 FabZAP ADC assay and Mole proliferationassay. Based on functional activity and diversity candidates wereselected for exploratory scale expression.

Strategy 2:

Candidates with human/cyno/mouse x-reactivity (5 HCDR3 families) wereaffinity matured and after IgG conversion candidates were selected forexpression.

In summary, 82 purified IgG candidates from strategy 1 and 2 weresubjected to in-depth characterization. From this pool of 82, 26 IgGcandidates were selected for upscaled production, toxin conjugation andsubsequent testing as antibody drug conjugates in in vitro and in vivoexperiments.

Upon in-depth characterization, the 26 antibodies (14 candidates fromstrategy 1 and 12 candidates from strategy 2) belonging to 16 differentHCDR3 families were selected for upscaled production and testing asantibody-DM1 conjugate. Candidates were selected according to followingcriteria: 1) Potent killing of wildtype and mutant cKIT expressing cellsin Fab-DM1 piggyback assay with EC50 in the sub- to low-nanomolar range,2) the KD values of 24/26 IgGs for cyno cKIT are within 3-fold range tothat determined for the human cKIT. In addition, 12/26 IgGs crossreactwith mouse and rat cKIT expressed on cells.

Selected candidates from this screening could be assigned to differentepitope bins:

1) 19/26 IgGs belong to Bin 1 or Bin 6 (binding to cKIT D1-3, ligandbinding domains)2) 6/26 IgGs belong to Bin 8 (binding to cKIT D4-5, dimerizationdomains)3) 1/26 IgGs belong to Bin 2, which had high affinity to human cKIT buthad only weak affinity to cyno cKIT. An example of an antibody to comefrom this type of screening protocol is antibody 20376.

Example 4 Constructs for Human, Cyno, Mouse and Rat cKIT ECD Proteins

Human, mouse and rat cKIT extracellular domains were gene synthesizedbased on amino acid sequences from the GenBank or Uniprot databases (seeTable 2 below). Cynomolgus cKIT and 1 ECD cDNA template were genesynthesized based on amino acid sequences information generated usingmRNA from various cyno tissues (e.g. Zyagen Laboratories; Table 2below). All synthesized DNA fragments were cloned into appropriateexpression vectors e.g. hEF1-HTLV based vector (pFUSE-mIgG2A-Fc2) withC-terminal tags to allow for purification.

TABLE 2 Accession SEQ ID Name Description Number NO HumanHuman cKIT tr. variant 2, residues 26-520-TAG NM_001093772 (SEQ IDcKIT D1-5 QPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLCTDPGFVK NO. 154)WTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQSSIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKEQIHP HTLFTPRSHHHHHH HumanHuman cKIT tr. Variant 1, residues 26-311-TAG NM_000222 (SEQ IDcKIT D1-3 QPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLCTDPGFVK NO. 155)WTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFM CYANNTFGSANVTTTLEVVDKGRSHHHHHHHuman Human cKIT tr. variant 1, residues 311-524-TAG NM_000222 (SEQ IDcKIT D4-5 GFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWI NO. 156)YMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQSSIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKGNNKEQIHPHTLFTPRSHHHHHH CynomolgusCynomolgus monkey cKIT, residues 25-520-TAG Not applicable. (see monkeybelow) cKIT D1-5 Mouse cKITMouse cKIT tr. variant 1, residues 26-527-TAG NM_001122733 (SEQ ID D1-5SQPSASPGEPSPPSIHPAQSELIVEAGDTLSLTCIDPDFV NO. 157)RWTFKTYFNEMVENKKNEWIQEKAEATRTGTYTCSNSNGLTSSIYVFVRDPAKLFLVGLPLFGKEDSDALVRCPLTDPQVSNYSLIECDGKSLPTDLTFVPNPKAGITIKNVKRAYHRLCVRCAAQRDGTWLHSDKFTLKVRAAIKAIPVVSVPETSHLLKKGDTFTVVCTIKDVSTSVNSMWLKMNPQPQHIAQVKHNSWHRGDFNYERQETLTISSARVDDSGVFMCYANNTFGSANVTTTLKVVEKGFINISPVKNTTVFVTDGENVDLVVEYEAYPKPEHQQWIYMNRTSANKGKDYVKSDNKSNIRYVNQLRLTRLKGTEGGTYTFLVSNSDASASVTFNVYVNTKPEILTYDRLINGMLQCVAEGFPEPTIDWYFCTGAEQRCTTPVSPVDVQVQNVSVSPFGKLVVQSSIDSSVFRHNGTVECKASNDVGKSSAFFNFAFKEQIQAHTLFTPLEVLFQGPRSPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWV ERNSYSCSVVHEGLHNHHTTKSFSRTPGKRat cKIT Rat cKIT, residues 25-526-TAG NM_022264 (SEQ ID D1-5SQPSASPGEPSPPSIQPAQSELIVEAGDTIRLTCTDPAFV NO. 158)KWTFEILDVRIENKQSEWIREKAEATHTGKYTCVSGSGLRSSIYVFVRDPAVLFLVGLPLFGKEDNDALVRCPLTDPQVSNYSLIECDGKSLPTDLKFVPNPKAGITIKNVKRAYHRLCIRCAAQREGKWMRSDKFTLKVRAAIKAIPVVSVPETSHLLKEGDTFTVICTIKDVSTSVDSMWIKLNPQPQSKAQVKRNSWHQGDFNYERQETLTISSARVNDSGVFMCYANNTFGSANVTTTLKVVEKGFINIFPVKNTTVFVTDGENVDLVVEFEAYPKPEHQQWIYMNRTPTNRGEDYVKSDNQSNIRYVNELRLTRLKGTEGGTYTFLVSNSDVSASVTFDVYVNTKPEILTYDRLMNGRLQCVAAGFPEPTIDWYFCTGAEQRCTVPVPPVDVQIQNASVSPFGKLVVQSSIDSSVFRHNGTVECKASNAVGKSSAFFNFAFKGNSKEQIQPHTLFTPRSLEVLFQGPGSPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLF ACSVVHEGLHNHLTTKTISRSLGK

TABLE 3 Sequences of cynomolgus cKIT proteinAmino acid sequence in one letter code, signal peptide SEQ ID Constructunderlined NO CynomolgusMYRMQLLSCIALSLALVTNSQPSVSPGEPSPPSIHPAKSELIVRVGNEIRLLC (SEQ IDmonkey cKIT IDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSSSIYV NO. 159)D1-5 FVRDPAKLFLVDRSLYGKEDNDTLVRCPLTDPEVTSYSLKGCQGKPLPKDLRFVPDPKAGITIKSVKRAYHRLCLHCSADQEGKSVLSDKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNASIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNASGPPFGKLVVQSSIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKGNN KEQIHPHTLFTPRSHHHHHHExpression of Recombinant cKIT Proteins

The desired cKIT recombinant proteins were expressed in HEK293 derivedcell lines (293FS) previously adapted to suspension culture and grown inserum-free medium FreeStyle-293 (Gibco, catalogue #12338018). Both smallscale and large scale protein production were via transient transfectionand was performed in multiple shaker flasks (Nalgene), up to 1 L each,with 293Fectin (Life Technologies, catalogue #12347019) as a plasmidcarrier. Total DNA and 293Fectin was used at a ratio of 1:1.5 (w:v). DNAto culture ratio was 1 mg/L. The cell culture supernatants wereharvested 3-4 days post transfection, centrifuged and sterile filteredprior to purification.

Example 5 Purification of Human, Cyno, Mouse and Rat cKIT ECD Protein,and of cKIT Subdomains 1-3, and 4-5 Tagged Protein Purification

Recombinant Fc-tagged cKIT extracellular domain proteins (e.g., humancKIT ECD-Fc, human cKIT (ECD subdomains 1-3, 4-5)-Fc, cyno cKIT-mFc, ratcKIT-mFc, mouse cKIT-mFc) were purified from the cell culturesupernatant. The clarified supernatant was passed over a Protein ASepharose column which had been equilibrated with PBS. After washing tobaseline, the bound material was eluted with Pierce Immunopure low pHElution Buffer, or 100 mM glycine (pH 2.7) and immediately neutralizedwith ⅛^(th) the elution volume of 1 M Tris pH 9. The pooled protein wasconcentrated if necessary using Amicon Ultra 15 mL centrifugalconcentrators with 10 kD or 30 kD nominal molecular weight cut-offs. Thepools were then purified by SEC using a Superdex 200 26/60 column toremove aggregates. The purified protein was then characterized bySDS-PAGE and SEC-MALLS (Multi-angle laser light scattering).Concentration was determined by absorbance at 280 nm, using thetheoretical absorption coefficients calculated from the sequence byVector NTI.

Example 6 Binding of cKIT Abs to cKIT ECD Subdomains

To help define the binding sites of the cKIT Abs, the human cKIT ECD wasdivided into subdomains 1-3 (ligand binding domain) and subdomains 4-5(dimerization domain). To determine which subdomains were bound, asandwich ELISA assay was employed. 1 ug/ml of ECD diluted in 1×Phosphate buffered saline corresponding to cKIT subdomains 1-3,subdomains 4-5 or full-length cKIT ECD were coated on 96 well Immulon4-HBX plates (Thermo Scientific Cat#3855, Rockford, Ill.) and incubatedovernight at 4° C. Plates were washed three times with wash buffer (1×Phosphate buffered saline (PBS) with 0.01% Tween-20 (Bio-Rad 101-0781)).Plates were blocked with 280 ul/well 3% Bovine Serum Albumin diluted in1×PB S for 2 hrs at room temperature. Plates were washed three timeswith wash buffer. Antibodies were prepared at 2 ug/ml in wash bufferwith 5-fold dilutions for 8 points and added to ELISA plates at 100ul/well in triplicate. Plates were incubated on an orbital shakershaking at 200 rpm for 1 hr at room temperature. Assay plates werewashed three times with wash buffer. Secondary antibody F(ab′)₂ FragmentGoat anti-human IgG (H+L) (Jackson Immunoresearch Cat#109-036-088, WestGrove, Pa.) was prepared 1:10,000 in wash buffer and added to ELISAplates at 100 ul/well. Plates were incubated with secondary antibody for1 hr at room temperature shaking at 200 rpm on an orbital shaker. Assayplates were washed three times with wash buffer. To develop the ELISAsignal, 100 ul/well of Sure Blue® TMB substrate (KPL Cat#52-00-03,Gaithersburg, Md.) was added to plates and allowed to incubate for 10mins at room temperature. To stop the reaction 50 ul of 1N HydrochloricAcid was added to each well. Absorbance was measured at 450 nM using aMolecular Devices SpectraMax M5 plate reader. To determine the bindingresponse of each antibody the optical density measurements wereaveraged, standard deviation values generated and graphed using Excel.The binding domains of each individual anti-cKIT antibody is found inTable 5 below.

Example 7 Affinity Measurements of cKIT Abs

Affinity of the antibodies to cKIT species orthologues and also to cKITwas determined using SPR technology using a Biacore® 2000 instrument (GEHealthcare, Pittsburgh, Pa.) and with CM5 sensor chips.

Briefly, HBS-P (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 0.005% SurfactantP20) supplemented with 2% Odyssey® blocking buffer (Li-Cor Biosciences,Lincoln, Nebr.) was used as the running buffer for all the experiments.The immobilization level and analyte interactions were measured byresponse unit (RU). Pilot experiments were performed to test and confirmthe feasibility of the immobilization of the anti-human Fc antibody(Catalog number BR100839, GE Healthcare, Pittsburgh, Pa.) and thecapture of the test antibodies.

For kinetic measurements, the experiments were performed in which theantibodies were captured to the sensor chip surface via the immobilizedanti-human Fc antibody and the ability of the cKIT proteins to bind infree solution was determined Briefly, 25 mg/ml of anti-human Fc antibodyat pH 5 was immobilized on a CM5 sensor chip through amine coupling atflow rate of 5 μl/minute on all two flow cells to reach 10,500 RUs.0.1-1 μg/ml of test antibodies were then injected at 10 μl/min for 1minute. Captured levels of the antibodies were generally kept below 200RUs. Subsequently, 3.125-50 nM of cKIT receptor extracellular domains(ECD) were diluted in a 2-fold series and injected at a flow rate of 40μl/min for 3 min over both reference and test flow cells. Table oftested ECDs is listed below. Dissociation of the binding was followedfor 10 min. After each injection cycle, the chip surface was regeneratedwith 3 M MgCl₂ at 10 μl/min for 30 s. All experiments were performed at25° C. and the response data were globally fitted with a simple 1:1interaction model (using Scrubber 2® software version 2.0b (BioLogicSoftware) to obtain estimates of on rate (k_(a)), off-rate (k_(d)) andaffinity (K_(D)).

TABLE 4 ECD isotype and source ECD Isotype Tag Source Human C-terminal6x His NVS Cyno C-terminal 6x His NVS Mouse C-terminal 6x His SinoBiological Inc (Catalog number: 50530-M08H) Rat C-terminal mFc NVS

Table 5 lists the domain binding and affinity. As shown in the Table,the antibodies 9p3, NEG024, NEG027, NEG085, NEG086, NEG087 and 20376 allreact with human cKIT at the nanomolar level, and have similaraffinities for those tested against cynomolgus monkey ECD. However, only20376 cross reacted with mouse. None of the antibodies testedcross-reacted with rat cKIT.

TABLE 5 Affinity human Affinity Affinity Affinity to Domain cKIT cynocKIT mouse cKIT rat cKIT Ab binding (nM) (nM) (nM) (nM) 9P3 d1-3 20 notNot reactive Not reactive determined NEG024 d1-3 1.31 1.15 Not reactiveNot reactive NEG026 d1-3 not not Not reactive Not reactive deter-determined mined NEG027 d1-3 1.34 not Not reactive Not reactivedetermined NEG085 d1-3 8.4 6.14 Not reactive Not reactive NEG086 d1-31.44 1.34 Not reactive Not reactive NEG087 d1-3 1.13 1.39 Not reactiveNot reactive 20376 d1-3 9.1 4.8  2.5 Not reactive

Example 8 Preparation of ADCs Preparation of the DM1 Conjugates byOne-Step Process

Individual cKIT antibodies were diafiltered into a reaction buffer (15mM potassium phosphate, 2 mM EDTA, pH 7.6) via Tangential FlowFiltration (TFF#1) prior to the start of the conjugation reaction.Subsequently, a cKIT antibody (about 5.0 mg/mL) was mixed with DM1(5.6-fold molar excess relative to the amount of antibody) and then withSMCC (about 5.0-fold excess relative to the amount of antibody). Thereaction was performed at 20° C. in 15 mM potassium phosphate buffer (pH7.6) containing 2 mM EDTA and 10% DMA for approximately 16 hours. Thereaction was quenched by adding 1 M acetic acid to adjust the pH to5.50. After pH adjustment, the reaction mixture was filtered through amulti-layer (0.45/0.22 μm) PVDF filter and purified and diafiltered intoa 20 mM succinate buffer (pH 5.0) containing 8.22% sucrose usingTangential Flow Filtration (TFF#2). An example of the instrumentparameters for the Tangential Flow Filtration are listed in Table 6below.

TABLE 6 Instrument parameters for the Tangential Flow Filtration TFFParameter TFF#1 Set Point TFF#2 Set Point Bulk Concentration (Cb - g/L)20 20 TMP (psi) 12-18 12-18 Feed Flow rate (LMH) 324  324  Membrane Load(g/m2) 110-150 110-150 Diavolumes 10 14 Diafiltration Buffer 15 mMpotassium 20 mM Succinate, phosphate, 2 mM 8.22% Sucrose, EDTA, pH 7.6pH 5.0 Temperature (° C.) RT (20-25) RT (20-25)

Conjugates obtained from the process described above was analyzed by: UVspectroscopy for cytotoxic agent loading (Maytansinoid to AntibodyRatio, MAR); SEC-HPLC for determination of conjugate monomer; andreverse-phase HPLC or hydrophobic shielded phase (Hisep)-HPLC for freemaytansinoid percentage.

Preparation of DM1 Conjugates by In Situ Process

The anti-cKIT antibodies can also be conjugated by an in situ processaccording to the following procedures. cKIT antibodies were conjugatedto DM1 using the sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) linker.Stock solutions of DM1 and sulfo-SMCC heterobifunctional linker wereprepared in DMA. Sulfo-SMCC and DM1 thiol were mixed together to reactfor 10 minutes at 25° C. in DMA containing 40% v/v of aqueous 50 mMsuccinate buffer, 2 mM EDTA, pH 5.0, at the ratio of DM1 to linker of1.3:1 mole equivalent and a final concentration of DM1 of 1.95 mM. Theantibody was then reacted with an aliquot of the reaction to give a moleequivalent ratio of SMCC to Ab of around 6.5:1 under final conjugationconditions of 2.5 mg/mL of Ab in 50 mM EPPS, pH 8.0 and 10% DMA (v/v).After approximately 18 hours at 25° C., the conjugation reaction mixturewas purified using a SEPHADEX™ G25 column equilibrated with 10 mMsuccinate, 250 mM glycine, 0.5% sucrose, 0.01% Tween 20, pH 5.5.

Either method is useful in the conjugation of antibodies. The Tablebelow provides an example of cKIT ADCs.

TABLE 7 Properties of DM1-conjugated antibodies Ab MAR Monomer (%) Yield(%) Free drug (%) 9P3 3.6 99 none detected NEG024 4 98 70 0.7 NEG026 498 71 1.2 NEG027 4 98 68 1.2 NEG085 3.5 99 88 0.7 NEG086 3.5 99 83 1.5NEG087 3.6 99 90 1.1 20376 3.8 99 84 none detectedPreparation of ADCs with the SPDB Linker

Anti-cKIT antibodies, for example, antibody 9P3, (8 mg/ml) were modifiedwith N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB, 5.0, 5.5 and 4.9fold molar excess respectively) for 120 minutes at 25° C. in 50 mMpotassium phosphate buffer (pH 7.5) containing 50 mM NaCl, 2 mM EDTA,and 5% DMA. The modified Ab without purification was subsequentlyconjugated to DM4 (1.7 fold molar excess over the unbound linker) at afinal modified antibody concentration of 4 mg/mL in 50 mM potassiumphosphate buffer (pH 7.5) containing 50 mM NaCl, 2 mM EDTA, and 5% DMAfor 18 hours at 25° C. The conjugation reaction mixture was purifiedusing a SEPHADEX™ G25 column equilibrated and eluted with 10 mMsuccinate, 250 mM glycine, 0.5% sucrose, 0.01% Tween 20, pH 5.5.

Preparation of ADCs with the CX1-1 Linker

Anti-cKIT antibodies, for example, antibody 9P3 (5.0 mg/mL) were mixedwith DM1 (7.15-fold molar excess relative to the amount of antibody) andthen with CX1-1 (5.5-fold excess relative to the amount of antibody).The reaction was performed at 25° C. in 60 mM EPPS[4-(2-Hydroxyethyl)-1-piperazinepropanesulfonic acid] buffer (pH 8.5)containing 2 mM EDTA and 5% DMA for approximately 16 hours. The reactionmixture was then purified using a SEPHADEX™ G25 column equilibrated andeluted in 10 mM succinate, 250 mM glycine, 0.5% sucrose, 0.01% Tween 20,pH 5.5.

An example comparing the in vitro efficacies of antibody-MCC-DM1,antibody-SPDB-DM4 and antibody-CX1-1-DM is shown in FIG. 2.

Example 9 Affinity of ADCs Relative to Parental Antibodies

The affinity of the antibodies to cKIT following conjugation to SMCC-DM1was determined using Biacore technology using a Biacore® T100 instrument(GE Healthcare, Pittsburgh, Pa.) and CM5 sensor chips using similarmethodology to that described in Example 7 above.

For the antibodies assessed, similar affinity estimates for binding tohuman cKIT were obtained for SMCC-DM1 conjugated antibodies relative toparental unconjugated antibodies, suggesting that conjugation does notappreciably impact antibody binding (Table 8).

TABLE 8 Affinities of unconjugated and MCC-DM1 conjugated antibodiesHuman c-Kit ECD (nM) Unconjugated -MCC-DM1 NEG024 1.3 1.1 NEG085 4.2 5.2NEG086 1.4 1.8 20376 9.1 11.2

Example 10 Activity of 9P3-MCC-DM1, 9P3-SPDB-DM4 and 9P3-CX1-1-DM1 on apanel of cell lines

Following conjugation to the MCC-DM1 linker-payload, the ability of theantibody drug conjugates (ADCs) to inhibit the proliferation of AML,SCLC, GIST, and melanoma cell lines was determined. The GIST-T1 cellline was generously provided by Dr. Takahiro Taguchi, Kochi U., Japan.The GIST430 and GIST882 cell lines were kindly provided by Dr. JonathanFletcher, Brigham and Women's Hospital, Boston, Mass.

For small cell lung cancer (SCLC), the NCI-H526 and the NCI-H1048 celllines were used. NCI-H526 is a high cKIT expressor and was obtained fromATCC (CRL-5811, ATCC Manassas, Va.). NCI-H1048 expresses cKIT at a lowerlevel, and was also obtained from the ATCC (CRL-5853). CMK-11-5 is anAML line that expresses high levels of cKIT ((JCRB Cat#IFO50430, Japan)see also Nagano et al., Int. J. Hematol. 1992; 56:67-78)). UKE-1 is alsoan AML cell line and it expresses low amounts of cKIT. The UKE-1 cellline was generously provided by Professor Walter Fiedler, UniversityHospital Eppendorf, Hamburg, Germany. Kasumi 1 was obtained from theATCC (CRL-2724). Kasumi-6 was obtained from the ATCC (CRL-2775).MDA-MB-453 were obtained from ATCC (HTB-131). NCI-H889 and NCI-H1930lines were purchased from ATCC (CRL-5817 and CRL-5906 respectively).Hel92.1.7 cells were obtained from Sigma-Aldrich (Cat#92111706-1VL,Sigma Aldrich, St. Louis, Mo.). The M-07e and SKNO1 cells were purchasedfrom DSMZ, ACC-104 and ACC-690 respectively (DSMZ, Braunschweig, Del.)The OCI-M1 cell line is also from DSMZ (ACC-529).

Briefly, cells were cultured in a tissue culture incubator at 37° C.with 5% CO₂ in culture medium as recommended by the supplier. On the dayof the assay, cells were washed twice with PBS (Cellgro, catalog#21-031-CV), prior to being treated with 0.1% trypsin-EDTA (in-housetechnical services) for 5 min and resuspended in the recommended culturemedium. Cells were then counted and seeded in 96 well plates (Costarcatalog #3603, Corning, Tewksbury, Mass.) at densities of 2,000-10,000cells/well in 100 μl of cell culture medium. A duplicate plate wasgenerated for a day 0 measurement and all plates were incubated in atissue culture incubator at 37° C. with 5% CO₂ overnight. Medium onlywells were also generated to act as negative controls. Following thisincubation, 100 μl/well of Cell titer Glo® reagent (Promega catalog#G7573, Madison, Wis.) was added to the day 0 plates, which were thenshaken gently for 2 min, incubated for 10 min, and the resultingluminescence intensity was measured using a Perkin Elmer WallacMicrobeta Trilux® plate reader (Perkin Elmer, Waltham, Mass.). Test ADCswere serially diluted to a 3× stock solution in the appropriate cellculture medium and 50 μl of 3× serially diluted ADCs were added (finalassay concentration 0.0002-68 nM DM1 equivalents) prior to incubation ina tissue culture incubator at 37° C. with 5% CO₂ for 5 days. Followingthis incubation period, relative cell viability was determined via theaddition of Cell titer Glo® reagent as described above. The effect ofthe ADCs on cell proliferation was calculated using the average of theduplicates as follows: (% Inhibition=(ADCtreated−untreated)/(untreated−Day 0)*100). The % inhibition data wasfitted to a 4-parameter logistic equation and GI₅₀ values weredetermined

As shown in FIG. 1, cKIT ADCs were tested in a proliferation assay on apanel of GIST (GIST T-1, GIST882, GIST430), SCLC (NCI-H526, NCI-H1048)and AML (Kasumi-6, Kasumi-1) cell lines. IC50 and maximum killing valuesare listed in the table. MDA-MB453 (breast cancer cell line) does notexpress cKIT. IgG-MCC-DM1 is the isotype control. As demonstrated byFIG. 1, all of the cKIT ADCs had nanomolar to sub-nanomolar IC50s in theseven lines used. This indicates that the cKIT ADCs have a broadspectrum of indications, and could be used wherever a tumor isexpressing appropriate levels of cKIT.

The ability of an anti-cKIT antibody (9P3) conjugated via the SPDB-DM4and CX1-1-DM1 linker-payload was also evaluated and is shown in FIG. 2.These studies, which were conducted as described above, revealed thatthe anti-cKIT ADC evaluated was also a potent inhibitor of cellproliferation using SPDB-DM4 or CX1-1-DM1, suggesting that their abilityto successfully deliver toxin to kill cells is not limited to MCC-DM1.FIG. 1 and FIG. 2 both provide for cKIT ADCs that are effective in thenanomolar to sub-nanomolar range.

In addition, FIG. 3 is a plot of anti-cKIT ADC GI50 against cKITreceptor level, and what indications (AML, GIST, melanoma and SCLC). Asshown in FIG. 3, anti-cKIT ADC is efficacious across all of the listedindications.

Example 11 In Vitro Activity of cKIT-MCC-DNB ADCs on GIST, SCLC and AMLCell Lines

Following conjugation to the MCC-DM1 linker-payload, the ability of theantibody drug conjugates (ADCs) to inhibit the proliferation of AML,SCLC and GIST cell lines was determined For a listing of the cells usedin these experiments by supplier see Example 10 above.

Briefly, cells were cultured in a tissue culture incubator at 37° C.with 5% CO₂ in culture medium as recommended by the supplier. On the dayof the assay, cells were washed twice with PBS (Cellgro, Cat #21-031-CV,Corning Tewksbury, Mass.), prior to being treated with 0.1% trypsin-EDTA(in-house technical services) for 5 min and resuspended in therecommended culture medium. Cells were then counted and seeded in 96well plates (Costar catalog #3603, Corning, Tewksbury, Mass.) atdensities of 5,000 cells/well for AML and SCLC cells and 10,000cells/well for GIST cells in 100 μl of cell culture medium. A duplicateplate was generated for a day 0 measurement and all plates wereincubated in a tissue culture incubator at 37° C. with 5% CO₂ overnight.Following this incubation, 100 μl/well of Cell titer Glo® reagent(Promega catalog #G7573, Promega, Madison, Wis.) was added to the day 0plates, which were then shaken gently for 2 min, incubated for 10 min,and the resulting luminescence intensity was measured using a PerkinElmer Wallac Microbeta® Trilux plate reader (Perkin Elmer, Waltham,Mass.). Test ADCs were serially diluted to a 3× stock solution in theappropriate cell culture medium and 50 μl of 3× serially diluted ADCswere added (final assay concentration of 0.0002-68 nM DM1 equivalents)prior to incubation in a tissue culture incubator at 37° C. with 5% CO₂for 5 to 8 days. Following this incubation period, relative cellviability was determined via the addition of Cell titer Glo® reagent asdescribed above. The effect of the ADCs on cell proliferation wascalculated using the average of the duplicates as follows: (% of maximumaffection (A_(MAX))=(untreated−highest ADC concentration treated)*100).

The % inhibition data was fitted to a 4-parameter logistic equation andGI₅₀ values were determined. This data is shown in FIGS. 4-9. As shownin the graphs, an IgG-MCC-DM1 conjugate is used as control. All of theADCs tested have greater activity than the control antibody. Asdemonstrated by the curves in FIGS. 4-9, the anti-cKIT ADCs, forexample, the NEG085, NEG024 and 20376 antibodies were very effective inreducing cell proliferation, and thus would efficacious in the treatmentof GIST, AML and SCLC.

Example 12 Ouantitation of cKIT Surface Receptor Density on Cell Linesby FACS (Fluoresence Activated Cell Sorting)

Quantum Simply Cellular Beads (Bangs Laboratories, Inc. Catalog #815,Fishers, Ind.) were used as standards. Antibody Binding Capacity of beadstandards range from 0 to about 310,000. Beads or five hundred thousandcells were centrifuged and washed two times with 100 μl/sample of FACSbuffer (PBS, 0.2% BSA, 0.1% NaAz). After each washing step, beads orcells were centrifuged and carefully re-suspended. Following washes,FACS buffer was added and 10 μg/ml of APC-Mouse Anti-Human CD117 (BDPharmigen Catalog #550412, BD Biosciences, San Jose, Calif.) or 10 μg/mlof APC-Mouse IgG κ Isotype Control (BD Pharmigen Catalog #554681) wasadded to the corresponding wells, for a final volume of 100 μl/sample.

The cell-antibody suspensions were then incubated on ice for 1 hour.Following incubation, cells were spun down and washed two times with 100μl FACS buffer. After each washing step, beads or cells were centrifugedand carefully re-suspended.

Non-viable cells were excluded by re-suspension in 100 μl/sample 7-AAD(BD Pharmigen Catalog #559925)-containing FACS buffer. Samples wereincubated on ice for 10 minutes and were analyzed in BD FACS Canto II®(BD Biosciences, San Jose, Calif.). Geomean of signal per sample wasdetermined using FlowJo® software, and antigen densities were determinedas described in the Quantum Simply Cellular manual. Analyses of in vitrocell line sensitivity to ADCs and cell line receptor density were donein TIBCO Spotfire 4.0.

This receptor density is shown on the Y-axis of FIG. 3. A receptordensity analysis is useful in this aspect as an initial biomarker forpatient stratification. For example, in FIG. 3, a high receptor densityis correlating with efficacy of the anti-cKIT ADC GI50 shown on theX-axis. Analysis of receptor density is useful in a clinical setting,for determining which patients should receive an anti-cKIT ADCtherapeutic.

Example 13 Epitope Mapping of cKIT to 9P3 Antibody by Deuterium ExchangeMass Spectrometry (HDx-MS)

Deuterium exchange mass spectrometry (HDx-MS) measures the deuteriumuptake on the amide backbone of a protein. These measurements aresensitive to the amide's solvent accessibility and to changes in thehydrogen bonding network of the backbone amides. HDx-MS is often used tocompare proteins in two different states, such as apo and ligand-bound,and coupled with rapid digestion with pepsin. In such experiments onecan locate regions, typically of 10 to 15 amino acids, that showdifferential deuterium uptake between two different states. Regions thatare protected are either directly involved in ligand binding orallosterically affected by binding of the antibody to the ligand.

In these experiments, the deuterium uptake of cKIT extra-cellular domain(SEQ ID NO:160, see below) was measured in the absence and presence of atherapeutic mAb, 9P3. Regions in cKIT that show a decrease in deuteriumuptake upon binding of the antibody are likely to be involved in theepitope; however, due to the nature of the measurement it is alsopossible to detect changes remote from the direct binding site(allosteric effects). Usually, the regions that have the greatest amountof protection are involved in direct binding although this may notalways be the case. In order to delineate direct binding events fromallosteric effects orthogonal measurements (e.g. X-ray crystallography,alanine mutagenesis) are necessary.

TABLE 9 cKIT extra-cellular domain construct SEQ ID NO: 160LENGTH: 503 amino acids TYPE: Protein ORGANISM: HumanQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLCTDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQSSIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKEQIHPHTLFTPRSHHHHHH

The cKIT epitope mapping experiments are performed on a Waters Synapt®G2 HDx-MS platform, which includes LEAP® robot system, nanoACQUITY® UPLCSystem, and Synapt® G2 mass spectrometer. In this method, triplicatecontrol experiments are carried out as follows. 300 pmol (1.4 mg/ml) ofcKIT antigen is diluted into 110 ul of 95% deuterated PBS buffer (pH7.4) and incubates at room temperature on a bench rotator for 25 minutes(% D=85.5%). Deuterium exchange is quenched by 1:1 dilution with coldquench buffer (6M Urea and 1M TCEP pH=2.5) on ice for 5 min. Afterquenching the tube is transferred onto a LEAP system (Thermo box is setat 2° C.) and the quenched sample is injected by the LEAP system ontothe UPLC system for analysis. The UPLC system incorporates animmobilized pepsin column 2.1 mm×30 mm (Life Technologies 2-3131-00)that is maintained at 12° C. An 8-minute 2 to 35% acetonitrile gradientand Waters UPLC CSH C18 1.0×100 mm column is used for separation. Next,triplicate experiments are carried out using the antibody. 300 pmol of9P3 antibody is immobilized on Protein G agarose beads (ThermoScientific Cat#22851) using standard techniques. Briefly, the antibodyis centrifuged to remove a storage buffer. Then 200 ul of PBS buffer (pH7.4) and 300 pmol of cKIT are added to the immobilized Ab and incubatefor 30 min at room temperature. After incubation, the complex iscentrifuged and washed with 200 ul PBS buffer and centrifuged again. Fordeuterium exchange, 200 ul of deuterated PBS is added to theantigen-antibody complex for incubation at room temperature for 25minutes (% D=85.5%). Deuterium buffer is then removed, and immediately,125 ul ice cold quench buffer is added. After quenching for 5 minutes,the column is centrifuged and the flow-through is transferred into aprechilled HPLC vial. The sample is analyzed using the same on-linepepsin digestion/LC-MS setup as the control experiment.

The results of these measurements are summarized in FIG. 10 and FIG. 11.FIG. 10 shows the baseline corrected differences between the control and9P3 antibody bound sample divided by the standard error in themeasurement. In this plot the more negative value indicates a greateramount of protection in a given region upon binding of 9P3 antibody tocKIT antigen. Upon binding of 9P3 to cKIT we observe the mostsignificant amounts of protection in the following two regions of cKIT:VFVRDPAKLFL ((Region 1, 109-119 (SEQ ID NO. 161)) and HCSDQEGKSLV((Region 2, 185-196 (SEQ ID NO.162)). Region 1 comprises residues109-119 and is part of the D1 and D2 domains. Region 2 comprisesresidues 185-196 and is part of the D2 domain. In FIG. 11, we havemapped the two most protected regions (see FIG. 10) onto the crystalstructure of cKIT extra-cellular domain (PDB ID 2e9w). In addition, wehave also labeled the SCF binding sites on cKIT as site I, II, and IIIusing literature values (Yuzawa et al., Cell 2007; 130: 323-334). Thereare two key findings from FIG. 11. First, regions 1 and 2 are very closetogether in the crystal structure even though they are far apart inprimary sequence space. This observation suggests that both couldpotentially be part of the epitope and if so, the epitope for 9P3 isdiscontinuous. Second, regions 1 and 2 are remote from the SCF bindingsites reported in literature. This is an important observation becauseit suggests that 9P3 antibody does not directly interfere with ligandbinding. Instead the antibody might sterically interfere with ligandbinding and/or with the dimerization of the receptor upon ligandbinding. In separate competition assays, using ELISA and FACS weobserved partial blocking of SCF binding to cKIT by 9P3 so there appearsto be partial steric interference. In conclusion, the HDx-MS dataindicate that the epitope for 9P3 antibody consists of a discontinuousepitope that is remote from the SCF binding sites. NEG024, NEG085,NEG086, NEG027 and NEG087 are expected to have the same mechanism ofaction.

Example 14 The Ability of cKIT ADCs to Act as (Monists was EvaluatedUsing a cKIT Wild Type Cell Line Mo7e and a cKIT Mutant Cell Line GISTT-1

To evaluate the potential agonistic properties of cKIT ADCs, 2×10⁶ ofGIST T-1 (kindly provided by Dr. Takahiro Taguchi, Kochi U., Japan) orMo7e (DSMZ, ACC-104) cells were serum starved overnight at 37° C. with5% CO₂ (DMEM for GIST T-1 and RPMI for Mo7e supplemented with 0.1% FBS)in 6-well plate (NUNC catalog #14067). Cells were treated with 10 ng/mlrh-SCF (R&D, Cat#255-SC, R&D, Berkeley, Calif.), 5 ug/ml NEG085-MCC-DM1,NEG024-MCC-DM1, and 20376-MCC-DM1 for 15 minutes at 37° C. One well wasdesignated as untreated (UT). Cells were harvested in 1 ml PBS. The cellpellets were lysed on ice for 60 mins in 30 ul lysis buffer: 20 mMTris-HCl; pH7.5, 137 mM NaCl, 1% Triton X-100, 15% Glycerol, proteaseand phosphatase inhibitors. Lysates were then spun down for 40 mins at12,000 rpm at 4° C. 20 μg of each sample was boiled for 10 min at 75° C.and loaded on a 12-well NuPAGE® 4-12% Bis-Tris gel (Life Technologies,NP0322BOX, Carlsbad, Calif.). After protein transfer to membrane blots,membranes were blocked in TBST-5% milk at room temperature for 1 hourand then probed with primary antibodies overnight at 4° C. Blots werewashed in TBST (4×5 mins) on the next day. Blots were incubated in thesecondary antibody (goat-anti rabbit-HRP 1:30,000, Santa Cruz) for 1 hrat room temperature. Blots were washed in TBST (4×5 mins) and developed.

The primary antibodies used for Western blotting were α-cKIT, Tyr703(Cell Signaling Technology Cat#3073, Beverly, Mass.), α-cKIT Tyr721(NOVUS, Cat#NBP1-51412, Novus, Littleton, Colo.), AKT Ser473 (CellSignaling Technology Cat#9271), AKT (Cell Signaling TechnologyCat#4691), ERK Thr202/Tyr204 (Cell Signaling Technology Cat#9101), ERK(Cell Signaling Technology Cat#9102), and GAPDH (Cell SignalingTechnology Cat#3683).

As shown in FIG. 12, the cKIT antibodies NEG085, NEG024 and 20376 canmediate phosphorylation of cKIT in the absence of ligand (SCF). However,downstream signaling pathways are not affected, as the signal does nottransduce to phospho ERK or phosphor AKT.

Example 15 cKIT Ab-Mediated Internalization of Surface cKIT on GIST-T1Cells as Determined by Flow Cytometry

The kinetics of cKIT antibody mediated internalization was evaluated bytreating with antibody in a cell monolayer using a temperature shiftmethod and flow cytometry readout. GIST-T1 (kindly provided by Dr.Takahiro Taguchi, Kochi U., Japan) cells were seeded at 2.5×10E5cells/well in five 12-well tissue culture treated plates (BD Falcon353043). The cells were incubated in a tissue culture incubator at 37°C. with 5% CO₂ overnight. The following day medium was removed andreplaced with 450 ul fresh medium. The cKIT antibodies NEG085, 20376 andan isotype control were prepared at 10×10 μg/ml concentration inappropriate cell culture medium and 50 μl of test cKIT antibody orisotype was added per well with final concentration of 10 ug/ml. Allcells were incubated for 1 hr on ice, followed by two washes with 1 mL1× Phosphate buffered saline (PBS) and resuspended in 500 μl cellculture medium. Plates #2-5 were transferred to 37° C. and harvested attime points; 30 min, 2 hr, 4 hr and 24 hr at 37° C. with 5% CO₂. 100 μlof cell dissociation buffer (Gibco Cat#13150-016, Life Technologies,Carlsbad, Calif.) was added to plate#1 (4° C. binding control) andincubated at 37° C. until cells were detached. Cells were neutralizedwith 100 μl of medium and transferred to a 96 well V-bottom tissueculture treated plate (Costar 3894). Cells were centrifuged and washedtwice with FACS Buffer (1× Phosphate Buffered Saline, 2% Fetal BovineSerum, 0.1% Sodium Azide). The Phycoerythrin conjugated goat anti-humanIgG secondary Ab (Invitrogen H10104, Life Technologies, Carlsbad,Calif.) was prepared at al to 100 ratio in FACS buffer. Secondaryantibody was added to cells at 100 μl/well and incubated with cells onice for 45 min. At the end of the incubation period, cells werecentrifuged and washed with FACS Buffer three times. Cells were fixedwith 100 μl/well of 1% paraformaldehyde and stored at 4° C. in the dark.Repeat cell disassociation, secondary antibody incubation and fixationsteps for the cells incubated at 37° C. for the various time points. Thefollowing day, all samples were analyzed using the BD FACSCanto II®equipment using a HTS system (BD Biosciences, San Jose, Calif.). Sampleswere analyzed with FlowJo software to obtain the Geometric Mean valuesof fluorescence for the Phycoerythrin channel. FIG. 13A is a plot of %of initial cell surface binding vs Geometric Mean-PE 4° C.binding/Geometric Mean-PE timepoint at 37° C.×100. As demonstrated inFIG. 13A, both antibodies NEG085 and 20376 bind cKIT on the cell surfaceand are rapidly internalized into the cell. This indicates that the cKITADCs disclosed would be rapidly internalized, thus delivering the toxininto the cell efficiently.

In another internalization experiment, the impact of NEG085 on cKitreceptor levels was evaluated on human bone marrow cells. Normal humanCD34+ bone marrow cells (All Cells, Cat #ABMO22F, Emeryville, Calif.)were thawed and washed with 10 mL of StemPro®-34 SFM medium (Gibco, LifeTechnologies, Carlsbad, Calif.). Cells were resuspended in 1.25 mL ofStemPro-34 SFM medium at 4×10⁵ cells/mL and split equally into twotubes. One tube was untreated, and the other was treated with 10 μg/mlof NEG085 and both were incubated at 37 C, 5% CO2. 100 μL of cellsuspension was collected at each timepoint (0, 15, 30, 60, 120, and 240min) from each condition, and placed into an ice cold collection tube tocease internalization. Cells were washed with 3 mL of ice-cold FBS stainbuffer and resuspended in 100 μL of FBS stain buffer. 5 mL of104D2-BV421 (mouse anti-human IgG1 k, Biolegend) was added to each tubeand incubated on ice for 1 hour. Following another wash with FBS stainbuffer, total cKit receptors were measured by flow cytometry byassessing the mean fluorescence intensity of BV421 on a FACS Canto II®(BD Biosciences, San Jose, Calif.).

As shown in FIG. 13B, cKIT is rapidly internalized upon binding ofNEG085, with the bulk of the internalization happening rapidly (15minutes) and then continuing to steadily decline the amount of cKIT onthe surface until the endpoint of 4 hours.

Example 16 Assessment of the Ability of NEG085-MCC-DM1 to Modulate cKITDegradation in a Wildtype cKIT Cell Line (NCI-H526) or a Mutant cKITCell Line (GIST-T1)

5×10⁶ of GIST-T1 (kindly provided by Dr. Takahiro Taguchi, Kochi U.,Japan) or NCI-H526 (ATCC CRL-5811) cells were seeded in growth media(DMEM, 10% FBS for GIST T-1 and RPMI, 10% FBS for NCI-H526) the nightbefore at 37° C. with 5% CO₂. Cells were then treated with 100 mMcycloheximide (CHX) (Cat#090M4009, Sigma-Aldrich, St.Louis, Mo.) inmethionine free medium (GIBCO: DMEM, 21013-024; RPMI, A14517-01, LifeTechnologies, Carlsbad, Calif.). Cells were either treated with 5 μg/mlADC (NEG085-MCC-DM1), 10 ng/ml rh-SCF (R&D, 255-SC), or both ADC andrh-SCF for 1, 4 or 6 hours at 37° C. with 5% CO₂. Cells were harvestedat 1 hour, 4 hour, and 6 hour post treatment in 1 ml PBS. The cellpellets were lysed on ice for 60 mins in 50 ul lysis buffer (20 mMTris-HCl; pH7.5, 137 mM NaCl, 1% Triton X-100, 15% Glycerol, proteaseand phosphatase inhibitors). Lysates were then spun down for 40 mins at12,000 rpm at 4° C. Five μg of each sample was boiled for 10 min at 75°C. and loaded on a 15-well NuPAGE® 4-12% Bis-Tris gel (NP0323BOX LifeTechnologies, Carlsbad, Calif.). After protein transfer to membraneblots, membranes were blocked in TBST-5% milk at room temperature for 1hour and then probed with anti cKIT antibody (Cell Signaling TechnologyCat#3074, Beverly, Mass.) overnight at 4° C. Blots were washed in TBST(4×5 mins) the next day. The blot was incubated in the secondaryantibody (goat-anti rabbit-HRP 1:30,000, Santa Cruz Biotechnologies,Dallas, Tex.) for 1 hour at room temperature. The blot was washed inTBST (4×5 mins) and developed. The primary antibodies used for Westernblotting were α-cKIT (Cell Signaling Technology Cat#3074) and GAPDH(Cell Signaling Technology Cat#3683). FIGS. 14 A/B show a timecourse ofcKIT receptor degradation mediated by NEG085-MCC-DM1. The degradationwas rapid with levels becoming very low/undetectable after 6 hours. Notethat the degradation of the cKIT receptor happens faster than SCF withNEG085-MCC-DM1 in the GIST T1 cells which express a mutant cKIT receptor(panel 14A). Also, the NEG085-MCC-DM1 does not block the cKIT receptorfrom binding SCF, as the addition of NEG085-MCC-DM1 and SCF provides forfaster degradation, as seen in FIG. 14B. If the NEG085-MCC-DM1 were aligand blocker, there would be no difference between NEG085-MCC-DM1 andNEG085-MCC-DM1 with SCF.

Example 17 Unconjugated NEG085 and 20376 do not Inhibit theProliferation of Mo7e, a SCF-Dependent Cell Line

To evaluate the potential antagonistic properties of the nakedantibodies and the ability of the antibody drug conjugates (ADCs) toinhibit the proliferation of a cKIT-expressing cell line, MO7e (DSMZ,Catalog #ACC-104, Braunschweig, Del.) were grown in the presence orabsence of cKIT ligand, Stem Cell Factor (SCF), for survival. MO7e cellswere grown in either 10 ng/ml human granulocyte-macrophagecolony-stimulating factor GM-CSF (R&D Systems Cat#215-GM, Minneapolis,Minn.) or 10 ng/ml human Stem Cell Factor SCF (R&D Systems Cat#255-SC)prior to seeding in 96 well plates (Costar Cat #3904, Corning,Tewksbury, Mass.) at 5000 cells/well in 100 μl dilution medium. Aduplicate plate was generated for a day 0 measurement and all plateswere incubated in a tissue culture incubator at 37° C. with 5% CO₂overnight. Following this incubation, an additional 50 μl of dilutionmedium was added, followed by 90 μl/well of Cell titer Glo® reagent(Promega Cat#G7573, Madison, Wis.) to each well of the designated “day0” plate. Assay plates were shaken gently for 20 min and the resultingluminescence intensity was measured using a Perkin Elmer 1450 MicrobetaTriLux® plate reader (Perkin Elmer, Waltham, Mass.). Test naked Abs andADCs were prepared at 3× concentration; 30 μg/ml in the appropriate cellculture medium and diluted serially 5-fold for 8 points. Medium onlywells were also generated to act as negative controls. 50 ul of 3×serially diluted antibodies or ADCs were added (final assayconcentration 0.0009-68 nM) prior to incubation in a tissue cultureincubator at 37° C. with 5% CO₂ for 5 days. Following this incubationperiod, relative cell viability was determined via the addition of Celltiter Glo reagent as described above. The effect of the ADCs on cellproliferation was calculated using the average of the duplicates asfollows: (% Inhibition=(ADC or Ab treated)/(untreated)*100) The %inhibition data was fitted to a 4-parameter logistic equation and IC₅₀values were determined

As shown in FIG. 15 and FIG. 16, the naked anti-cKIT antibodies do notinhibit cell proliferation. In FIG. 15, the NEG085-MCC-DM1 is comparedwith unconjugated NEG085, NEG024 and 20376. As shown clearly in thegraph, NEG085-MCC-DM1 inhibits cell proliferation of M07e cells at a lowconcentration, while the unconjugated antibodies do not have thiseffect. The IgG-MCC-DM1 control has a greater anti-proliferative effectthan unconjugated NEG085, NEG024 or 20376.

This is also seen in FIG. 16, where the experiment uses GM-CSF ratherthan SCF to negate the internalization effect on the cKIT receptor thatthe SCF ligand has. The result in FIG. 16 is consistent with that ofFIG. 15, that an unconjugated NEG085 antibody has no detrimental effecton cell proliferation, similar to an unconjugated IgG control. Insummary, the results shown on FIGS. 15 and 16 indicate that thereduction in cell proliferation is due to the conjugation of theanti-cKIT antibodies with the toxin.

Example 18 Evaluation of ADCC Activity In Vitro

The ability of the unconjugated anti-cKIT antibodies (NEG085, 20376) tomediate antibody dependent cellular cytotoxicity was determined versusUke-1 cells (target cells; generously provided by Professor WalterFiedler, University Hospital Eppendorf, Hamburg, Germany) inco-incubation with NK3.3 cells (killer cells or effector cells; kindlyprovided by Jacky Kornbluth from Saint Louis University). In brief,Uke-1 cells were stained with Calcein acetoxy-methyl ester (Calcein-AM;Sigma-Aldrich catalog #17783-5MG, St. Louis, Mo.), washed twice,pipetted into a 96-well microtiterplate (96 well, U-bottomed, clearplastic; Corning Costar, catalog #650 160, Tewksbury, Mass.) at aconcentration of 5000 cells per well and pre-incubated for 10 min with aserial dilution of the above mentioned antibodies and proteins (from50,000 to 0.003 μg per ml) before adding the effector NK3.3 cells for 1hour in an effector to target ratio of 20 to 1. In order to calculatethe antibody specific lysis of the target cells, a parallel incubationof target cells only without antibody or effector cells served as abaseline and negative control, whereas the positive control or maximallysis or hundred percent specific lysis was determined by lysis oftarget cells only with a 1% Triton-X 100 solution. As an additionalpositive control, MabCampath® (Sanofi, Paris, FR) was used, recognizingCD52 on the Uke-1cells. Following a co-incubation of target and effectorcells, the microtiterplate was centrifuged and an aliquot of thesupernatant fluid was transferred to another microtiterplate (96 well,flat-bottomed, black with clear bottom; Corning Costar, catalog #3904,Tewksbury, Mass.) and the concentration of free Calcein in solution wasdetermined with a fluorescence counter (Victor 3® multilabel counter,Perkin Elmer, Waltham, Mass.).

Results are presented in FIG. 17. Antibody Dependent Cell MediatedCytotoxicity (ADCC) is a mechanism of cell mediated immunity, whereby aneffector cell lyses a target cell that has been bound by specificantibodies. In this experiment, MabCampath® as well as the anti-cKITantibodies 20376 and NEG085 are unconjugated human IgG1 antibodies. Asshown in FIG. 17, only the MabCampath antibody mediated ADCC killing ofthe target cells. Both 20376 and NEG085 were not able to induce ADCCeven at higher concentrations. As such, any cell killing seen when oneof the ADCs is used, for example, NEG085-MCC-DM1, is not due to an ADCCmechanism of action.

Example 19 The Ability of NEG085 and 20376 to Cause Mast Cell Apoptosiswas Investigated Using Primary Human Mast Cells

Primary human mast cells were cultured from peripheral human bloodaccording to the methods described by Saito et al., Nature Protocols2006; 1(4):2178-2183. Mast cells, which had been in liquid culture for aminimum of one week, were incubated with increasing concentrations(0.05-100 nM) of the anti-human cKIT Abs, NEG085 and 20376, or anisotype control IgG, in the presence of 1.6 nM rhSCF (Genscript, Cat#Z00400, Piscataway, N.J.), for 48 h at 37° C. before the addition ofthe Caspase-Glo® 3/7 reagent (Promega, Cat#G8093, Madison, Wis.) tomeasure apoptosis. Following 30 min incubation at RT, luminescence wasrecorded on the BioTek® Synergy plate reader (BioTek, Winooski, Vt.).

As cKIT is expressed on mast cells, any therapeutic anti cKIT antibodiesshould not cause depletion of mast cells. FIG. 18 shows an apoptosisassay with primary human mast cells following treatment with eitheranti-human cKIT Abs or an isotype control Ab, in the presence of 1.6 nMrhSCF. Primary human mast cells were incubated with increasingconcentrations of the anti cKIT antibodies, NEG085 and 20376, or anisotype control IgG. As seen in FIG. 18, both the NEG085 and 20376unconjugated antibodies do not lead to apoptosis of human primary mastcells ex vivo.

Example 20 The Ability of NEG085 and 20376 to Mediate Mast CellDegranulation was Determined Using Primary Human Mast Cells

Primary human mast cells were cultured from peripheral human bloodaccording to the methods described by Saito et al., (supra). Mast cells,which had been in liquid culture for a minimum of one week, werepre-treated with 5% Ag-specific IgE JW8 (in-house batch ACE 27283), 95%non-specific monoclonal human IgE (Abbiotec, Cat #12030635, San Diego,Calif.) and 10 ng/mL rhIL-4 (R&D Systems Cat #204-IL, Minneapolis,Minn.) for 5 days at 37° C. The cells were then incubated withincreasing concentrations (0.05-100 nM) of an isotype control IgG, theanti-human cKIT Abs, 20376 and NEG085, the anti-IgE Ab, LE27, or theNIP(5)BSA antigen, in the presence of a goat anti-human IgG (H+L)Fc-specific Ab (Jackson ImmunoResearch, Cat #109-005-008-JIR, WestGrove, Pa.) for 90 min at 37° C. Cells were then centrifuged and thesupernatants were transferred into 96-well black-walled plates prior tothe addition of the β-hexosaminidase substrate. Following 90 minincubation at 37° C., the reaction was stopped by the addition oftris-base (Sigma, Cat #T1503-500G, pH 12, St. Louis, Mo.) and thefluorescence intensity was recorded on the Envision® plate reader.

As in the previous experiment in Example 19, it is important to assessany detrimental effect of anti-cKIT antibodies on mast cells. Where theprevious experiment examined apoptosis of mast cells, here theexperiments are directed to mast cell degranulation. As shown in FIG.19, the positive controls NIP(5) and LE27 show high levels of mast celldegranulation. In contrast, anti-cKIT antibodies NEG085 and 20376 do notinduce mast cell degranulation of human primary mast cells ex vivo.

Example 21 In Vivo on-Target Pharmacodynamic Marker Modulation by cKITADCs

Studies were conducted to assess the ability of the cKIT ADCNEG027-MCC-DM1 to modulate pharmacodynamic markers in vivo, including anexamination of the co-localization of NEG027 antibody to thepharmacodynamics (PD) event of mitotic arrest in the mutant cKITexpressing GIST T1 tumor xenograft. The goal of these studies was toevaluate the degree and duration of G2/M cell cycle arrest.

Presence of ADC was indirectly estimated by detecting human IgG antibody(which is NEG027 in the mouse) in the tumor using an immunohistochemicalapproach. An affinity purified rabbit anti-human IgG (H+L) was obtainedfrom Jackson ImmunoResearch Laboratories (Cat#309-005-082, West Grove,Pa.). The antibody reacts with whole molecule human IgG and the lightchains of other human immunoglobulins with minimal cross-reaction tomouse serum proteins. Briefly, the IHC protocol included heat andstandard exposure to Ventana Cell Conditioning #1 antigen retrievalreagent. The primary antibody was diluted to a working concentration of2 ug/ml and incubated for 32 minutes at room temperature. Subsequently,incubation with Ventana UltraMap pre-diluted HRP-conjugated anti-rabbitantibody (Cat #760-4315, Ventana, Tucson, Ariz.) was performed for 32minutes.

Accumulation of pHH3 positive nuclei, as assessed byimmunohistochemistry, was used as a marker of G2/M arrest. A rabbitpolyclonal antibody produced by immunizing animals with a syntheticphosphopeptide corresponding to residues surrounding Ser10 of humanhistone H3 (pHH3) was obtained from Cell Signaling Technology (Danvers,Mass., Cat#9701). Briefly, the IHC protocol included heat and standardexposure to Ventana Cell Conditioning #1 antigen retrieval reagent. Theprimary antibody was diluted to 1:50 and incubated for 60 minutes atroom temperature. Subsequently, incubation with Jackson ImmunoResearchLaboratories goat anti-rabbit biotinylated secondary antibody(Cat#111-065-144, West Grove, Pa.) was performed for 32 minutes.

To assess anti-cKIT ADC induced PD marker changes in the GIST T1subcutaneous tumor xenograft model, female SCID-beige mice wereimplanted subcutaneously with 10×10⁶ cells in a suspension containing50% Matrigel™ (BD Biosciences) in Hank's balanced salt solution. Thetotal injection volume containing cells in suspension was 200 μl. Micewere randomly assigned to receive a single i.v. dose of eitherNEG027-MCC-DM1 (2.5 mg/kg), non-specific IgG1-MCC-DM1 isotype control(2.5 mg/kg) or tris-buffered saline (TBS; 5 ml/kg) once tumors reachedbetween 300 and 500 mm³ (n=3/group). Immunostaining for human IgG showswhere NEG027 is located and this correlates with areas of a greaterdensity of pHH3 immunostaining (representative images shown in FIG. 20,providing support for colocalization of the antibody with thepharmacodynamic effect. Consistent with the expected mechanism of actionof the maytansinoid payload, NEG027-MCC-DM1 yielded a marked,time-dependent increase in the percentage of cells positive for pHH3positivity, peaking at 33 and 48 h post dose relative to thenon-specific isotype IgG1-MCC-DM1 or PBS treated controls, with signalback to baseline at around a week (representative images shown in FIG.21, graph shown in FIG. 22). Time dependent changes in cleaved caspase 3were also evaluated. In these studies, a rabbit polyclonal antibodyproduced by immunizing animals with a synthetic peptide corresponding toamino-terminal residues adjacent to (Asp175) in human caspase-3 wasobtained from EMD Millipore (Cat#PC679). The IHC protocol included Heatand Standard exposure to Ventana Cell Conditioning #1 antigen retrievalreagent. The primary antibody was diluted to 20 ug/ml and incubated for32 minutes at room temperature. Subsequently, incubation with JacksonImmunoResearch Laboratories goat anti-rabbit biotinylated secondaryantibody (Cat#111-065-144, West Grove, Pa.) was performed for 32minutes.

Similar to pHH3, time dependent changes in cleaved caspase 3 were alsoobserved (representative images shown in FIG. 21, graph shown in FIG.22). These data demonstrate that the cKIT ADC NEG027-MCC-DM1 is capableof eliciting robust in vivo cellular PD effects consistent with themechanism of action of the maytansinoid payload.

A representative photo of cKIT immunostaining on the GIST T1 tumor isshown to visualize the staining pattern in this xenograft model (FIG.21). A rabbit polyclonal antibody produced by immunizing animals with asynthetic peptide corresponding to amino acids 963 to 976 at thecytoplasmic c-terminal part of cKIT was obtained from Dako (Cat#A4502).Briefly, the IHC protocol included heat and standard exposure to VentanaCell Conditioning #1 antigen retrieval reagent. The primary antibody wasdiluted to a working concentration of 14 ug/ml and incubated for 60minutes at room temperature. Subsequently, incubation with VentanaUltraMap pre-diluted HRP-conjugated anti-rabbit antibody (Cat #760-4315)was performed for 16 minutes.

Example 22 In Vivo Efficacy of Anti-cKIT ADCs Against GastrointestinalStromal Tumor (GIST) in Mice

The anti-tumor activity of anti-cKIT ADCs was evaluated in several tumorxenograft models. The dose dependent antitumor activity andpharmacokinetics (PK) of a non-mouse cKIT cross-reactive anti-human cKITADC NEG027-MCC-DM1 was evaluated in the mutant cKIT expressing GIST T1subcutaneous tumor xenograft model. Female SCID-beige mice wereimplanted subcutaneously with 10×10⁶ cells containing 50% Matrigel™ (BDBiosciences) in Hank's balanced salt solution. The total injectionvolume containing cells in suspension was 200 μl.

Mice were enrolled in the study 10 days post implantation with averagetumor volume of 207 mm³. After being randomly assigned to one of fivegroups (n=9/group), mice were administered a single i.v. dose of TBS,the ADC vehicle (5 ml/kg), a non-specific isotype control IgG1-MCC-DM1(2.5 mg/kg), or NEG027-MCC-DM1 (0.625, 1.25 or 2.5 mg/kg). Tumor volumesand body weights were measured twice weekly. The control IgG1-MCC-DM1was not significantly active at 2.5 mg/kg. NEG027-MCC-DM1 at 0.625showed statistically significant efficacy compared to the TBS treatedgroup, however 1.25 and 2.5 mg/kg induced even greater efficacy, bothinducing similar tumor volume stasis as per caliper measurements,although a histological assessment did not show presence of tumor cells.Instead a mixture of connective tissue, adipose tissue and segments ofperipheral nerves and striated muscle were the main tissue components inthese sections. This supports a histological regression in the tumor(FIGS. 23-26).

From this study serum was also collected at 1 hour, 24 hours and 4, 7,11 and 21 days post-dose to measure antibody/ADC concentration over timeusing an anti-human IgG1 ELISA and an anti-DM ELISA, respectively. Toassess PK parameters, serum was collected via retro-orbital bleeds andanalyzed via ELISA. The total antibody PK assay measures total antibodyconcentration, with/without DM1 by colorimetric ELISA. Plates are coatedwith anti-human IgG (Fc specific), and detection is with anti-humanIgG-HRP before being read on an appropriate plate-reader. The conjugatePK assay measures antibody that is bound to at least 1 DM1 molecule bycolorimetric ELISA. In this format, plates are coated withanti-maytansine antibody, and detected with anti-human IgG-HRP. PK isdose proportional with an approximate serum half-life of seven days(FIGS. 23-24).

Since a single 0.625 mg/kg dose of NEG027-MCC-DM1 only caused GIST T1tumor growth delay, thus providing a dynamic range to assess differingADC activities, this dose level was selected to assess efficacy of a setof closely related ADCs, also derived from the original murine9P3-MCC-DM1 ADC. Female SCID-beige mice were implanted subcutaneouslywith 10)(10⁶ cells containing 50% Matrigel™ (BD Biosciences, San Jose,Calif.) in Hank's balanced salt solution. The total injection volumecontaining cells in suspension was 200 μl. Mice were enrolled in thestudy 10 days post implantation with average tumor volume of 195 mm³.After being randomly assigned to groups (n=8/group), mice wereadministered a single i.v. dose of TBS (8 ml/kg), a non-specific isotypecontrol IgG1-MCC-DM1 (10 mg/kg), NEG085-MCC-DM1 (0.625 mg/kg),NEG086-MCC-DM1 (0.625 mg/kg), NEG087-MCC-DM1 (0.625 mg/kg),NEG024-MCC-DM1 (0.625 mg/kg), or NEG026-MCC-DM1 (0.625 mg/kg). Tumorvolumes and body weights were measured twice weekly (FIG. 27-29). Thecontrol IgG1-MCC-DM1 even at the high dose of 10 mg/kg was not active.The anti-cKIT ADCs dosed at 0.625 mg/kg were not statistically differentfrom each other. NEG085-MCC-DM1 and NEG024-MCC-DM1 treated groups hadthe smallest tumor volumes in the tightest range.

From this study serum was also collected at 1 hour, 24 hours and 3, 7,10, 14 and 21 days post-dose to measure antibody/ADC concentration overtime using an anti-human IgG1 ELISA and an anti-DM ELISA, respectively.To assess PK parameters, serum was collected via retro-orbital bleedsand analyzed via ELISA. The total antibody PK assay measures totalantibody concentration, with/without DM1 by colorimetric ELISA. Platesare coated with anti-human IgG (Fc specific), and detection is withanti-human IgG-HRP before being read on an appropriate plate-reader. Theconjugate PK assay measures antibody that is bound to at least 1 DM1molecule by colorimetric ELISA. In this format, plates are coated withanti-maytansine antibody, and detected with anti-human IgG-HRP. TheseADCs showed similar serum exposures (FIG. 30).

Example 23 In Vivo Efficacy of Anti-cKIT ADCs Against Small Cell LungCancer in Mice

Antitumor activity of a set of ADCs were assessed in the NCI-H1048 smallcell lung cancer xenograft model with moderate cKIT immunostaining thatexhibits greater heterogeneity compared to GIST T1 tumor xenografts(FIG. 21-FIG. 30). NEG085-MCC-DM1 was compared to a set of cKIT ADCsthat are strong antagonists of cKIT signaling, none of which bind tomouse cKIT. Female SCID-beige mice were implanted subcutaneously with10×10⁶ cells containing 50% Matrigel™ (BD Biosciences) in Hank'sbalanced salt solution. The total injection volume containing cells insuspension was 200 μl. Mice were enrolled in the study 15 days postimplantation with average tumor volume of about 120 mm³. All treatedgroups received a single intravenous dose of 2 mg/kg. After beingrandomly assigned to groups (n=8/group), mice were administered a singlei.v. dose of TBS (5 ml/kg), a non-specific isotype control IgG1-MCC-DM1(2 mg/kg), NEG024-MCC-DM1, NEG085-MCC-DM1, and NEG086-MCC-DM1. Tumorvolumes and body weights were measured twice weekly (FIG. 31, 32). Thecontrol IgG1-MCC-DM1 was not active. NEG085-MCC-DM1 trended towardefficacy with a low AT/AC of 9%, but was not statistically differentfrom the vehicle at this 2 mg/kg dose. NEG024-MCC-DM1 and NEG026-MCC-DM1were significantly efficacious.

Antitumor efficacy of cKIT ADCs were also NCI-H1048 small cell lungcancer xenograft model, dose dependent antitumor activity ofNEG085-MCC-DM1 was assessed. Female SCID-beige mice were implantedsubcutaneously with 10×10⁶ cells containing 50% Matrigel™ (BDBiosciences) in Hank's balanced salt solution. The total injectionvolume containing cells in suspension was 200 μl. Mice were enrolled inthe study 11 days post implantation with average tumor volume of about150-200 mm³. After being randomly assigned to groups (n=8/group), micewere administered a single i.v. dose of TBS (5 ml/kg), a non-specificisotype control IgG1-MCC-DM1 (10 mg/kg), or NEG085-MCC-DM1 (2.5, 5 and10 mg/kg). Tumor volumes and body weights were measured twice weekly(FIG. 33, 34). The control IgG1-MCC-DM1 was not active, nor was the 2.5mg/kg dose of NEG085-MCC-DM1. However, the 5 and 10 mg/kg doses weresignificantly efficacious.

Antitumor activity of two anti-cKIT ADCs were assessed in a second smallcell lung cancer xenograft model with higher cKIT levels, similar to theGIST T1 tumor xenografts (representative photos on FIG. 21 and graphs inFIG. 35). Female SCID-beige mice were implanted subcutaneously with6×10⁶ cells containing 50% Matrigel™ (BD Biosciences) in Hank's balancedsalt solution. The total injection volume containing cells in suspensionwas 200 μl. Mice were enrolled in the study 6 days post implantationwith average tumor volume of about 150 mm³ After being randomly assignedto groups (n=9/group), mice were administered a single i.v. dose of TBS(8 ml/kg), a non-specific isotype control IgG1-MCC-DM1 (10 mg/kg),NEG024-MCC-DM1 (2.5, 5 and 10 mg/kg) and a mouse cross-reactive ADC20376-MCC-DM1 (10 mg/kg). Tumor volumes and body weights were measuredtwice weekly (FIG. 35 and FIG. 36). The control IgG1-MCC-DM1 was notactive. 20376-MCC-DM1 at 10 mg/kg initially regressed tumors, howeverafter the initial regression, tumor recurrence was seen. Significantdose dependent efficacy was observed with the three doses ofNEG024-MCC-DM1, with sustained long term regression at 10 mg/kg, withtumors starting to regrow after 60 days, suggesting 20376-MCC-DM1 mayrequire more than the single dose administered in this study. The serumexposure of a 10 mg/kg dose of 20376-MCC-DM1 and NEG024-MCC-DM1 wereabout equivalent.

Example 24 In Vivo Efficacy of Anti-cKIT ADCs Against Acute MyelogenousLeukemia in Mice

The dose dependent antitumor activity of anti-cKIT ADC murine9P3-MCC-DM1 and 9P3-SPDB-DM4 was evaluated in the mutant cKIT expressingacute myelogenous leukemia Kasumi-1 subcutaneous tumor xenograft model.Female SCID-beige mice were transplanted subcutaneously with 2-3 piecesof 1 mm³ fragmented Kasumi-1 tumor tissues on the right flank withMatrigel™ (BD Biosciences). Mice with Kasumi-1 tumors were enrolled inthe study 21 days post implantation with average tumor volume of 150mm³. After being randomly assigned to one of eight groups (n=8/group),mice were administered a single i.v. dose of PBS (200 ul), anon-specific isotype control IgG1-SPDB-DM4 (10 mg/kg), 9P3-SMCC-DM1 (10mg/kg) and 9P3-SPDB-DM4 (1 or 5 mg/kg). Tumor volumes and body weightswere measured three times weekly (FIG. 37). The control IgG1-SPDB-DM4was not significantly active at 10 mg/kg. Tumor growth regression wasobserved with 9P3-SPDB-DM4 at 5 mg/kg and 10 mg/kg doses.

TABLE 10 Kasumi-1 Efficacy Tumor Response Mean change Host Response oftumor Percent volume vs body control weight loss Survival Drug DoseSchedule (ΔT/ΔC) (%) (%) (Survivors/total) PBS  0 mg/kg single dose 1006.36 8/8 IV IgG-SPDB- 10 mg/kg single dose 98 3.06 8/8 DM4 IV 9P3-MCC-10 mg/kg single dose 11 −0.62 8/8 DM1 IV 9P3-SPDB-  1 mg/kg single dose65 −0.05 8/8 DM4 IV 9P3-SPDB-  5 mg/kg single dose −83 −1.72 8/8 DM4 IV

Example 25 In Vivo Efficacy of Anti-cKIT ADCs Against Mastocytosis inMice

The antitumor activity of anti-cKIT ADC murine 9P3-MCC-DM1 and9P3-SPDB-DM4 was evaluated in the mutant cKIT expressing HMC-1.2subcutaneous tumor xenograft model. The HMC-1.2 cell line was kindlyprovided by Dr. Joseph Butterfield, Mayo Clinic, Rochester, Minn. FemaleFoxn-1 nude mice were implanted subcutaneously with 3, 5, and 10×10⁶cells containing 50% Matrigel™ (BD Biosciences) in FBS-free DMEM media.The total injection volume containing cells in suspension was 100 μl.

HMC-1.2 tumor bearing mice in this study were enrolled 33 days postimplantation with average tumor volume of 100 mm³. After being randomlyassigned to one of three groups (n=4/group), mice were administered asingle i.v. dose of PBS (200 μl), 9P3-MCC-DM1 (10 mg/kg) or 9P3-SPDB-DM4(10 mg/kg). Tumor volumes and body weights were measured three timesweekly (FIG. 38). Tumor regression was observed at 9P3-SPDB-DM4 and9P3-SMCC-DM1 at 10 mg/kg.

TABLE 11 HMC-1 Study Tumor Response Mean change Host Response of tumorPercent volume vs body control weight loss Survival Drug Dose Schedule(ΔT/ΔC) (%) (%) (Survivors/total) PBS  0 mg/kg single dose 100 13.49 4/4IV 9P3-MCC- 10 mg/kg single dose 6 7.71 4/4 DM1 IV 9P3-SPDB- 10 mg/kgsingle dose −40 3.25 4/4 DM4 IV

Example 26 In Vivo Efficacy of a Mouse Cross-Reactive cKITADC20376-MCC-DM1

The dose dependent antitumor activity and pharmacokinetics (PK) of themouse cKIT cross-reactive anti-human cKIT ADC 20376-MCC-DM1 wasevaluated in the mutant cKIT expressing GIST T1 subcutaneous tumorxenograft model. Female SCID-beige mice were enrolled in the study 10days post implantation with average tumor volume of about 200 mm³. Afterbeing randomly assigned to one of five groups (n=9/group), mice wereadministered a single i.v. dose of TBS (5 ml/kg), a non-specific isotypecontrol IgG1-MCC-DM1 (10 mg/kg), NEG085-MCC-DM1 (0.625 mg/kg) or20376-MCC-DM1 (0.625, 2.5, 5 or 10 mg/kg). Tumor volumes and bodyweights were measured twice weekly (FIGS. 39-41). The controlIgG1-MCC-DM1 was not significantly active at 10 mg/kg. 203786-MCC-DM1was also ineffective, while NEG085-MCC-DM1 at 0.625 showed what may be ahint of efficacy, although also not statistically significant.20376-MCC-DM1 at 2.5, 5 and 10 mg/kg were all significantly efficacious.

From this study serum was also collected at 1 hour, 24 hours and 4, 7,11 and 21 days post-dose to measure antibody/ADC concentration over timeusing an anti-human IgG1 ELISA and an anti-DM ELISA, respectively. Toassess PK parameters, serum was collected via retro-orbital bleeds andanalyzed via ELISA. The total antibody PK assay measures total antibodyconcentration, with/without DM1 by colorimetric ELISA. Plates are coatedwith anti-human IgG (Fc specific), and detection is with anti-humanIgG-HRP before being read on an appropriate plate-reader. The conjugatePK assay measures antibody that is bound to at least 1 DM1 molecule bycolorimetric ELISA. In this format, plates are coated withanti-maytansine antibody, and detected with anti-human IgG-HRP. With themouse cKIT cross reactive ADC 20376-MCC-DM1 the PK was not doseproportional due to the ADC binding mouse cKIT in normal tissuesaffecting the exposure (tissue mediated drug disposition), and thusthere is a clear difference in serum concentrations between20376-MCC-DM1 and the non-mouse cKIT cross-reactive ADC NEG085-MCC-DM1(FIG. 40). This accounts for the difference in efficacy between the twoADCs at the low dose of 0.625 mg/kg. At the higher doses, the tissuemediated drug disposition effect is less pronounced and efficacy becomesapparent in the GIST T1 tumor xenograft model in mice.

Example 28 In Vivo Efficacy of cKIT ADCs with the SPDB-DM4Linker/Payload Against Gastrointestinal Stromal Tumors

The dose dependent antitumor activity of the murine 9P3 ADCs (from whichNEG024 and NEG085 were derived) with the MCC-DM1 (non-cleavable) andSPDB-DM4 (cleavable) linkers/payloads was compared in the mutant cKITexpressing GIST T1 subcutaneous tumor xenograft model. Female SCID-beigemice were enrolled in the study 18 days post implantation with averagetumor volume of about 170 mm³. After being randomly assigned to groups(n=8/group), mice were administered a single i.v. dose of TBS (5 ml/kg),unconjugated murine 9P3 antibody (10 mg/kg), a non-specific isotypecontrol IgG1-MCC-DM1 (5 mg/kg), non-specific isotype controlIgG1-MCC-DM1 (5 mg/kg), non-specific isotype control IgG1-SPDB-DM4 (10mg/kg), 9P3-MCC-DM1 (5 and 10 mg/kg or 9P3-SPDB-DM4 (2.5 and 5 mg/kg.Tumor volumes and body weights were measured twice weekly (FIGS. 42,43). Neither the control non-specific IgG1 ADCs nor the unconjugated 9P3were efficacious. All the 9P3. ADCs were efficacious at the tested doselevels; however, tumors from the 2.5 mg/kg 9P3-SPDB-DM4 treated groupappeared slightly less effective than the other groups.

The dose dependent antitumor activity of the murine 9P3 ADCs (from whichNEG024 and NEG085 were derived) with the MCC-DM1 (non-cleavable) andSPDB-DM4 (cleavable) linkers/payloads was compared in a second tumorxenograft model of mutant cKIT expressing gastrointestinal stromaltumor, GIST430. Female SCID-beige mice were enrolled in the study 11days post implantation with average tumor volume of about 200 mm³. Afterbeing randomly assigned to groups (n=9/group), mice were administered asingle i.v. dose of TBS (5 ml/kg), unconjugated murine 9P3 antibody (10mg/kg), a non-specific isotype control IgG1-MCC-DM1 (5 mg/kg),non-specific isotype control IgG1-MCC-DM1 (10 mg/kg), non-specificisotype control IgG1-SPDB-DM4 (5 mg/kg), 9P3-MCC-DM1 (10 mg/kg) or9P3-SPDB-DM4 (5 mg/kg). Tumor volumes and body weights were measuredtwice weekly (FIGS. 44, 45). Neither control non-specific IgG1 ADC wasefficacious. However, both 9P3 ADCs were similar efficacious at thetested dose levels.

Example 29 Formulation

The clinical service form (CSF) of the ADC is a lyophilisate in vialcontaining 50 mg anti-cKIT-MCC-DM1, 16.2 mg sodium succinate, 410.8 mgsucrose and 1 mg polysorbate 20 (without considering the overfill of 10%to allow for withdrawal of the declared content). After reconstitutionof the lyophilizate with 5 mL water for injection, a solution containing10 mg/mL anti-cKIT-MCC-DM1, 20 mM sodium succinate, 240 mM Sucrose and0.02% polysorbate 20 at a pH of 5.0 is obtained.

For subsequent intravenous administration, the obtained solution willusually be further diluted into a carrier solution to the ready-to-useADC solution for infusion.

For the CSF, an ADC concentration of 10 mg/mL was chosen based onpreliminary stability testing. A sucrose concentration of 240 mM wasselected in order to create an isotonic formulation, to maintain anamorphous lyophilizate cake structure and to afford proteinstabilization.

Important stability-indicating analytical methods to select the moststable formulation encompassed, amongst others, size-exclusionchromatography to determine aggregation levels, subvisible particulatematter testing, free Toxin determination and potency testing.

The pre-screening study showed that polysorbate 20 at a concentration of0.02% provides sufficient stabilization against mechanical stress. Theliquid and lyophilized stability studies at real-time and acceleratedstability conditions (25° C. and 40° C.) demonstrated that a succinatepH 5.0 formulation provides the overall best storage stability. Mostnotably in this formulation the best balance of all tested formulationsbetween aggregation and release of the free Toxin could be met. Afterthree months at 40° C. no noteworthy increase in degradation productscould be determined

It is understood that the examples and aspects described herein are forillustrative purposes only and that various modifications or changes inlight thereof will be suggested to persons skilled in the art and are tobe included within the spirit and purview of this application and scopeof the appended claims.

What is claimed is:
 1. An antibody drug conjugate of the formulaAb-(L-(D)_(n))_(n) or a pharmaceutically acceptable salt thereof;wherein Ab is an antibody or antigen binding fragment thereof thatspecifically binds to an epitope of human cKIT; L is a linker; D is adrug moiety; m is an integer from 1 to 8; and n is an integer from 1 to10.
 2. The antibody drug conjugate of claim 1, wherein said n is 3 or 4.3. The antibody drug conjugate of claim 1, wherein said antibody orantigen binding fragment thereof specifically binds the extracellulardomain of cKIT (SEQ ID NO.160).
 4. The antibody drug conjugate of claim1, wherein said antibody or antigen binding fragment specifically bindsto an epitope of human cKIT at domains 1-3 (SEQ ID NO:155).
 5. Theantibody drug conjugate of claim 1, wherein said antibody or antigenbinding fragment thereof specifically binds human cKIT at SEQ ID NO. 161or SEQ ID NO.162.
 6. The antibody drug conjugate of claim 1, whereinsaid antibody or antigen binding fragment thereof comprises a heavychain variable region that comprises: (a) a VH CDR1 of SEQ ID NO: 3, (b)a VH CDR2 of SEQ ID NO: 4, (c) a VH CDR3 of SEQ ID NO: 5; and a lightchain variable region that comprises: (d) a VL CDR1 of SEQ ID NO: 12,(e) a VL CDR2 of SEQ ID NO: 13, and (f) a VL CDR3 of SEQ ID NO:
 14. 7.The antibody drug conjugate of claim 1, wherein said antibody or antigenbinding fragment thereof comprises a heavy chain variable region thatcomprises: (a) a VH CDR1 of SEQ ID NO: 22, (b) a VH CDR2 of SEQ ID NO:23, (c) a VH CDR3 of SEQ ID NO: 24; and a light chain variable regionthat comprises: (d) a VL CDR1 of SEQ ID NO: 31, (e) a VL CDR2 of SEQ IDNO: 32, and (f) a VL CDR3 of SEQ ID NO:
 33. 8. The antibody drugconjugate of claim 1, wherein said antibody or antigen binding fragmentthereof comprises a heavy chain variable region that comprises: (a) a VHCDR1 of SEQ ID NO: 40, (b) a VH CDR2 of SEQ ID NO: 41, (c) a VH CDR3 ofSEQ ID NO: 42; and a light chain variable region that comprises: (d) aVL CDR1 of SEQ ID NO: 49, (e) a VL CDR2 of SEQ ID NO: 50, and (f) a VLCDR3 of SEQ ID NO:
 51. 9. The antibody drug conjugate of claim 1,wherein said antibody or antigen binding fragment thereof comprises aheavy chain variable region that comprises: (a) a VH CDR1 of SEQ ID NO:58, (b) a VH CDR2 of SEQ ID NO: 59, (c) a VH CDR3 of SEQ ID NO: 60; anda light chain variable region that comprises: (d) a VL CDR1 of SEQ IDNO: 67, (e) a VL CDR2 of SEQ ID NO: 68, and (f) a VL CDR3 of SEQ ID NO:69.
 10. The antibody drug conjugate of claim 1, wherein said antibody orantigen binding fragment thereof comprises a heavy chain variable regionthat comprises: (a) a VH CDR1 of SEQ ID NO: 76, (b) a VH CDR2 of SEQ IDNO: 77, (c) a VH CDR3 of SEQ ID NO: 78; and a light chain variableregion that comprises: (d) a VL CDR1 of SEQ ID NO: 85, (e) a VL CDR2 ofSEQ ID NO: 86, and (f) a VL CDR3 of SEQ ID NO:
 87. 11. The antibody drugconjugate of claim 1, wherein said antibody or antigen binding fragmentthereof comprises a heavy chain variable region that comprises: (a) a VHCDR1 of SEQ ID NO: 94, (b) a VH CDR2 of SEQ ID NO: 95, (c) a VH CDR3 ofSEQ ID NO: 96; and a light chain variable region that comprises: (d) aVL CDR1 of SEQ ID NO: 103, (e) a VL CDR2 of SEQ ID NO: 104, and (f) a VLCDR3 of SEQ ID NO:
 105. 12. The antibody drug conjugate of claim 1,wherein said antibody or antigen binding fragment thereof comprises aheavy chain variable region that comprises: (a) a VH CDR1 of SEQ ID NO:112, (b) a VH CDR2 of SEQ ID NO: 113, (c) a VH CDR3 of SEQ ID NO: 114;and a light chain variable region that comprises: (d) a VL CDR1 of SEQID NO: 121, (e) a VL CDR2 of SEQ ID NO: 122, and (f) a VL CDR3 of SEQ IDNO:
 123. 13. The antibody drug conjugate of claim 1, wherein saidantibody or antigen binding fragment thereof comprises a heavy chainvariable region that comprises: (a) a VH CDR1 of SEQ ID NO: 130, (b) aVH CDR2 of SEQ ID NO: 131, (c) a VH CDR3 of SEQ ID NO: 132; and a lightchain variable region that comprises: (d) a VL CDR1 of SEQ ID NO: 139,(e) a VL CDR2 of SEQ ID NO: 140, and (f) a VL CDR3 of SEQ ID NO: 141.14. The antibody drug conjugate of claim 1, wherein said linker isselected from the group consisting of a cleavable linker, anon-cleavable linker, a hydrophilic linker, a procharged linker and adicarboxylic acid based linker.
 15. The antibody drug conjugate of claim14, wherein the linker is derived from a cross-linking reagent selectedfrom the group consisting ofN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl4-(2-pyridyldithio)butanoate (SPDB),N-succinimidyl-4-(2-pyridyldithio)-2-sulfo-butanoate (sulfo-SPDB),N-succinimidyl iodoacetate (SIA),N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), maleimide PEG NHS,N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC),N-sulfosuccinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate(sulfo-SMCC) or 2,5-dioxopyrrolidin-1-yl17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazaheptadecan-1-oate(CX1-1).
 16. The antibody drug conjugate of claim 15, wherein saidlinker is derived from the cross-linking reagent N-succinimidyl4-(maleimidomethyl)cyclohexanecarboxylate (SMCC).
 17. The antibody drugconjugate of claim 16, wherein the cytotoxic agent is a maytansinoid.18. The antibody drug conjugate of claim 17, wherein the maytansinoid isN(2′)-deacetyl-N(2′)-(3-mercapto-1-oxopropyl)-maytansine (DM1) orN(2′)-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4).19. An antibody drug conjugate of the formula

or a pharmaceutically acceptable salt thereof; wherein Ab is an antibodyor antigen binding fragment thereof that specifically binds to humancKIT, and comprises at least n number of primary amines; and n is aninteger from 1 to
 10. 20. The antibody drug conjugate of claim 19,wherein said Ab is an antibody or antigen binding fragment thereofcomprises a heavy chain variable region that comprises: (a) a VH CDR1 ofSEQ ID NO: 3, (b) a VH CDR2 of SEQ ID NO: 4, (c) a VH CDR3 of SEQ ID NO:5; and a light chain variable region that comprises: (d) a VL CDR1 ofSEQ ID NO: 12, (e) a VL CDR2 of SEQ ID NO: 13, and (f) a VL CDR3 of SEQID NO:
 14. 21. The antibody drug conjugate of claim 19, wherein said Abis an antibody or antigen binding fragment thereof comprises a heavychain variable region that comprises: (a) a VH CDR1 of SEQ ID NO: 22,(b) a VH CDR2 of SEQ ID NO: 23, (c) a VH CDR3 of SEQ ID NO: 24; and alight chain variable region that comprises: (d) a VL CDR1 of SEQ ID NO:31, (e) a VL CDR2 of SEQ ID NO: 32, and (f) a VL CDR3 of SEQ ID NO: 33.22. The antibody drug conjugate of claim 19, wherein said Ab is anantibody or antigen binding fragment thereof comprises a heavy chainvariable region that comprises: (a) a VH CDR1 of SEQ ID NO: 40, (b) a VHCDR2 of SEQ ID NO: 41, (c) a VH CDR3 of SEQ ID NO: 42; and a light chainvariable region that comprises: (d) a VL CDR1 of SEQ ID NO: 49, (e) a VLCDR2 of SEQ ID NO: 50, and (f) a VL CDR3 of SEQ ID NO:
 51. 23. Theantibody drug conjugate of claim 19, wherein said Ab is an antibody orantigen binding fragment thereof comprises a heavy chain variable regionthat comprises: (a) a VH CDR1 of SEQ ID NO: 58, (b) a VH CDR2 of SEQ IDNO: 59, (c) a VH CDR3 of SEQ ID NO: 60; and a light chain variableregion that comprises: (d) a VL CDR1 of SEQ ID NO: 67, (e) a VL CDR2 ofSEQ ID NO: 68, (f) a VL CDR3 of SEQ ID NO:
 69. 24. The antibody drugconjugate of claim 19, wherein said Ab is an antibody or antigen bindingfragment thereof comprises a heavy chain variable region that comprises:(a) a VH CDR1 of SEQ ID NO: 76, (b) a VH CDR2 of SEQ ID NO: 77, (c) a VHCDR3 of SEQ ID NO: 78; and a light chain variable region that comprises:(d) a VL CDR1 of SEQ ID NO: 85, (e) a VL CDR2 of SEQ ID NO: 86, and (f)a VL CDR3 of SEQ ID NO:
 87. 25. The antibody drug conjugate of claim 19,wherein said Ab is an antibody or antigen binding fragment thereofcomprises a heavy chain variable region that comprises: (a) a VH CDR1 ofSEQ ID NO: 94, (b) a VH CDR2 of SEQ ID NO: 95, (c) a VH CDR3 of SEQ IDNO: 96; and a light chain variable region that comprises: (d) a VL CDR1of SEQ ID NO: 103, (e) a VL CDR2 of SEQ ID NO: 104, and (f) a VL CDR3 ofSEQ ID NO:
 105. 26. The antibody drug conjugate of claim 19, whereinsaid Ab is an antibody or antigen binding fragment thereof comprises aheavy chain variable region that comprises: (a) a VH CDR1 of SEQ ID NO:112, (b) a VH CDR2 of SEQ ID NO: 113, (c) a VH CDR3 of SEQ ID NO: 114;and a light chain variable region that comprises: (d) a VL CDR1 of SEQID NO: 121, (e) a VL CDR2 of SEQ ID NO: 122, and (f) a VL CDR3 of SEQ IDNO:
 123. 27. The antibody drug conjugate of claim 19, wherein said Ab isan antibody or antigen binding fragment thereof comprises a heavy chainvariable region that comprises: (a) a VH CDR1 of SEQ ID NO: 130, (b) aVH CDR2 of SEQ ID NO: 131, (c) a VH CDR3 of SEQ ID NO: 132; and a lightchain variable region that comprises: (d) a VL CDR1 of SEQ ID NO: 139,(e) a VL CDR2 of SEQ ID NO: 140, and (f) a VL CDR3 of SEQ ID NO: 141.28. (canceled)
 29. The antibody drug conjugate of claim 19, wherein saidn is 3 or
 4. 30. The antibody drug conjugate of claim 19, wherein saidantibody is a human antibody.
 31. The antibody drug conjugate of claim19, wherein said antibody is a monoclonal antibody.
 32. The antibodydrug conjugate of claim 19, wherein said linker is selected from thegroup consisting of a cleavable linker, a non-cleavable linker, ahydrophilic linker, a procharged linker and a dicarboxylic acid basedlinker.
 33. The antibody drug conjugate of claim 32, wherein the linkeris derived from a cross-linking reagent selected from the groupconsisting of N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl4-(2-pyridyldithio)butanoate (SPDB),N-succinimidyl-4-(2-pyridyldithio)-2-sulfo-butanoate (sulfo-SPDB),N-succinimidyl iodoacetate (SIA),N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), maleimide PEG NHS,N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC),N-sulfosuccinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate(sulfo-SMCC) or 2,5-dioxopyrrolidin-1-yl17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazaheptadecan-1-oate(CX1-1).
 34. The antibody drug conjugate of claim 33, wherein saidlinker is N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate(SMCC).
 35. The antibody drug conjugate of claim 19, wherein said drugmoiety is selected from a group consisting of a V-ATPase inhibitor, apro-apoptotic agent, a Bcl2 inhibitor, an MCL1 inhibitor, a HSP90inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubulestabilizer, a microtubule destabilizer, an auristatin, a dolastatin, amaytansinoid, a MetAP (methionine aminopeptidase), an inhibitor ofnuclear export of proteins CRM1, a DPPIV inhibitor, proteasomeinhibitors, inhibitors of phosphoryl transfer reactions in mitochondria,a protein synthesis inhibitor, a kinase inhibitor, a CDK2 inhibitor, aCDK9 inhibitor, a kinesin inhibitor, an HDAC inhibitor, a DNA damagingagent, a DNA alkylating agent, a DNA intercalator, a DNA minor groovebinder and a DHFR inhibitor.
 36. The antibody drug conjugate of claim35, wherein the cytotoxic agent is a maytansinoid.
 37. The antibody drugconjugate of claim 36, wherein the maytansinoid isN(2′)-deacetyl-N(2′)-(3-mercapto-1-oxopropyl)-maytansine (DM1) orN(2′)-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4).38. A pharmaceutical composition comprising the antibody drug conjugateof claim 1 and a pharmaceutically acceptable carrier.
 39. Thepharmaceutical composition of claim 38 wherein said composition isprepared as a lyophilisate.
 40. The pharmaceutical composition of claim39, wherein said lyophilisate comprises the antibody drug conjugate ofclaim 1, sodium succinate, and polysorbate
 20. 41. A method of treatingan cKIT positive cancer in a patient in need thereof, comprisingadministering to said patient the antibody drug conjugate of claim 1.42. The method of claim 41, wherein said cancer is selected from thegroup consisting of gastrointestinal stromal tumors (GIST), small celllung cancer (SCLC), acute myelod leukemia (AML), melanoma, mast cellleukemia (MCL), mastocytosis, neurofibromatosis, breast cancer,non-small cell lung cancer (NSCLC) and pancreatic cancer.
 43. (canceled)44. (canceled)
 45. The antibody or antigen binding fragment of claim 1,wherein said antibody or antigen binding fragment is a single chainantibody (scFv).
 46. A nucleic acid that encodes the antibody or antigenbinding fragment of claim
 1. 47. A vector comprising the nucleic acid ofclaim
 46. 48. A host cell comprising the vector according to claim 47.49. A process for producing an antibody or antigen binding fragmentcomprising cultivating the host cell of claim 48 and recovering theantibody from the culture.
 50. A process for producing an anti-cKITantibody drug conjugate comprising: (a) chemically linking SMCC to adrug moiety DM-1; (b) conjugating said linker-drug to the antibodyrecovered from the cell culture of claim 49; and (c) purifying theantibody drug conjugate.
 51. The antibody drug conjugate made accordingto claim 50 having an average maytansinoid to antibody ratio (MAR),measured with a UV spectrophotometer, about 3.5.
 52. A diagnosticreagent comprising the antibody or antigen binding fragment thereof ofclaim 1 which is labeled.
 53. The diagnostic reagent of claim 52,wherein the label is selected from the group consisting of a radiolabel,a fluorophore, a chromophore, an imaging agent, and a metal ion.